Factor H binding proteins (fHbp) with altered properties and methods of use thereof

ABSTRACT

Factor H binding proteins that can elicit antibodies that are bactericidal for at least one strain of  N. meningitidis , and methods of use of such proteins, are provided.

CROSS REFERENCE

This application is a divisional of U.S. patent application Ser. No.15/792,519, now U.S. Pat. No. 10,342,860, which claims the benefit ofcontinuation U.S. patent application Ser. No. 13/074,957, now U.S. Pat.No. 9,827,300, which claims the benefit of U.S. Provisional PatentApplication Nos. 61/319,181, filed Mar. 30, 2010, 61/334,542, filed May13, 2010, 61/381,025, filed Sep. 8, 2010, 61/423,757, filed Dec. 16,2010, and 61/440,227, filed Feb. 7, 2011, each of which applications isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant nos. RO1 AI046464, RO1 AI 082263, and AI 070955 awarded by the National Instituteof Allergy and Infectious Diseases, National Institutes of Health. Thegovernment has certain rights in the invention.

INTRODUCTION

Neisseria meningitidis is a Gram-negative bacterium which colonizes thehuman upper respiratory tract and is responsible for worldwide sporadicand cyclical epidemic outbreaks of, most notably, meningitis and sepsis.The attack and morbidity rates are highest in children under 2 years ofage. Like other Gram-negative bacteria, Neisseria meningitidis typicallypossess a cytoplasmic membrane, a peptidoglycan layer, an outer membranewhich together with the capsular polysaccharide constitute the bacterialwall, and pili, which project into the outside environment. Encapsulatedstrains of Neisseria meningitidis are a major cause of bacterialmeningitis and septicemia in children and young adults. The prevalenceand economic importance of invasive Neisseria meningitidis infectionshave driven the search for effective vaccines that can confer immunityacross different strains, and particularly across genetically diversegroup B strains with different serotypes or serosubtypes.

Factor H Binding Protein (fHbp, also referred to in the art aslipoprotein 2086 (Fletcher et al (2004) Infect Immun 72:2088-2100),Genome-derived Neisserial antigen (GNA) 1870 (Masignani et al. (2003) JExp Med 197:789-99) or “741”) is an N. meningitidis protein which isexpressed in the bacterium as a surface-exposed lipoprotein. Animportant function of fHbp is to bind human complement factor H (fH),which down-regulates complement activation. Binding of fH to thebacterial surface is an important mechanism by which the pathogensurvives in non-immune human serum or blood and evades innate hostdefenses. Recently, genetic variation in the human factor H gene clusterwas found to affect susceptibility to developing meningococcal disease(Davila S et al. (2010) Nat Genetics doi:10.1038/ng.640). Binding of fHto fHbp is specific for human fH and could account for why Neisseriameningitidis is strictly a human pathogen.

There remains a need for a fHbp polypeptide that can elicit effectivebactericidal antibody responses.

SUMMARY

Factor H binding proteins that can elicit antibodies that arebactericidal for at least one strain of N. meningitidis, and methods ofuse of such proteins, are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Panel A, Standard curve of human fH concentration as measured byELISA with meningococcal fHbp as the antigen in the wells. See Example 1for details. Panel B, Human fH concentrations in sera of human fHtransgenic (Tg) mice, which encompasses human fH-negative littermates ofTg mice or known wildtype BALB/c mice, and the human fH concentrationsin the sera of humans. See Example 1.

FIG. 2. Serum IgG antibody responses of human fH transgenic (fH Tg)BALB/c mice and wildtype (WT) BALB/c mice immunized with a meningococcalgroup C conjugate control vaccine (Panels A and B), and serumbactericidal titers against group C strain 4243 (Panel C). The conjugatevaccine does not bind human fH. See Example 1 for details. Panel D.Human fH binds to the wild-type fHbp vaccine, but does not bind to thecontrol MenC-CRM conjugate vaccine or to certain mutant fHbp vaccines,shown schematically to accompany Table 5 in the example section. Mouse(or rabbit, or rat, etc.) fH does not bind to wildtype fHbp.

FIG. 3. Relationships between serum human fH concentrations of fHtransgenic mice and serum bactericidal antibody responses to vaccinationwith wild-type fHbp that binds human fH (panel A) or to vaccination withR41S mutant that does not human fH (panel B). Panel C shows the GMTratios (mutant/wild-type vaccine in relationship to serum human fHconcentrations of immunized fH transgenic mice) estimated from thegeneral linear regression model. See Example 4 for details.

FIG. 4. Binding of human fH, and anti-fHbp mAbs, JAR 4, and JAR 5, bywild-type and mutant fHbps (mutants of fHbp ID1 containing Glu to Alasubstitutions) as measured by enzyme-linked immunosorbent assay (ELISA).

FIG. 5. SDS-PAGE size and purity analysis of WT fHbp ID 1 and a doublemutant of ID 1, E218A/E239A. The molecular masses in kDa are indicatedon the left.

FIG. 6. Soluble fHbp inhibition of anti-fHbp MAb binding to immobilizedwild-type fHbp by ELISA.

FIGS. 7A-7D. FIGS. 7A and 7B depict differential scanning calorimetry offHbp ID 1 wildtype and E218A/E239A double mutant protein (FIG. 7A) andof fHbp ID 1 wildtype and R41S mutant protein (FIG. 7B). FIG. 7C depictsanti-fHbp IgG antibody titers of mice immunized with fHbp ID 1 wildtypeor E218A/E239A double mutant protein determined by ELISA. IgG Anti-fHbpantibody responses of mice immunized with WT or mutant fHbp. In Study 3,mice were immunized with three doses of recombinant WT or mutant fHbpadsorbed with Freund's Adjuvant (FA) or aluminum hydroxide (Al(OH)₃); inStudy 4, CD-1 mice were immunized with one dose of WT or mutant fHbpadsorbed with aluminum hydroxide (Al(OH)₃); in Study 5, BALB/c mice wereimmunized with three doses of WT or mutant fHbp adsorbed with aluminumhydroxide (Al(OH)₃). Shaded bars, WT fHbp; open bars, E218A/E239A mutantfHbp. FIG. 7D depicts anti-fHbp IgG titers of BALB/c mice that weregiven two doses of fHbp vaccine in Study 6.

FIG. 8. Panel A depicts binding of fH to natural fHbp variants. Wells ofmicrotiter plates were coated with recombinant fHbps representingvariants fHbp IDs 1, 14, or 15. Binding of human fH was measured asdescribed in the Examples section. Panels B and C depict binding of thevariants fHbp IDs 1, 14, and 15 to MAb JAR4 and JAR5, respectively.

FIG. 9A. Structure of the complex between fHbp and a fragment of humanfH. fHbp is shown on the bottom in black with fH shown at the top ingrey in cartoon representation. Structural model of fHbp bound to afragment of fH based on published atomic coordinates (Schneider et al.((2009) Nature 458:890-3)). The black ribbons represent the respectiveN- and C-terminal domains of the fHbp molecule. The gray ribbonrepresents the sixth and seventh short consensus repeat domains of humanfH previously shown to mediate the interaction of human fH and fHbp(Schneider et al. ((2009) Nature 458:890-3). The zoomed-in view on theleft focuses on the arginine residue at position 41, showing a chargedH-bond with fH, which was predicted to be eliminated when arginine wasreplaced by serine (right lower inset). The figure was generated usingMacPyMol (www.pymol.org).

FIG. 9B shows the amino acid sequence of human factor H (fH), which isalso known as GenBank accession no. NP_000177 (P08603), and its encodingnucleic acid as NM_000186.

FIG. 10. Binding of fH (panel A) or anti-fHbp MAbs JAR 4 (panel B) orJAR 5 (panel C) to R41S and R41A mutants of fHbp ID 1, as measured byELISA. Binding of human fH (panel A), and anti-fHbp MAbs (panels B andC) was measured as described in Example 2.

FIG. 11. Binding of human factor H (left column) or anti-fHbp MAb JAR 4(right column) to different fHbps in variant group 1 and theircorresponding R41S mutants. Binding was measured as described in Example2. Panels A and B show the binding results for fHbp ID 4. Panels C and Dshow the binding results for fHbp ID 9. Panels E and F show the bindingresults for fHbp ID 74. “ID” refers to fHbp amino acid sequence variantidentification (ID) number, as described in the Neisseria Multi LocusSequence Typing website (hypertext transferprotocol)://pubmlst(dot)org/neisseria/fHbp/.

FIG. 12. Binding of human fH and control anti-fHbp MAbs to fHbp andcorresponding R41S mutants of fHbps in variant group 2. Panels A and Bshow binding of human factor H to wildtype (WT) fHbp ID 19 and R41Smutant of fHbp ID 19. Panels C and D show binding of human factor H toWT fHbp ID 22 and R41S mutant of fHbp ID 22. Panels E and F show bindingof human factor H to WT fHbp ID 77 and R41S mutant of fHbp ID 77. TheMAb controls were JAR 4 (Panels B and D) or JAR 11 (Panel F).

FIG. 13. Effect of serum anti-fHbp antibody elicited in human fHtransgenic mice on fH binding to fHbp. Binding of fH to fHbp wasmeasured by ELISA in 1:100 dilutions of pre-immunization (panel A,pre-immune) and post-immunization (panel B, post-immune) sera fromindividual transgenic mice immunized with wild-type fHbp ID 1 or R41Smutant ID 1 fHbp vaccines. For the aluminum control groups, the opensquares represent data from serum pools from transgenic mice whose seracontain human fH and the closed triangles represent data from sera fromwild-type mice whose sera do not contain human fH. The OD valuesrepresent the quantity of bound human fH as detected with sheepanti-human fH and donkey anti-sheep IgG conjugated to alkalinephosphatase. Panel C, IgG anti-fHbp titers in post-immunization serashowing similar antibody responses to both vaccines. Panel D, Inhibitionof binding of human fH to fHbp in the presence of added human fH. PanelE, Relationship of percent inhibition of fH binding and SBA titers ofhuman fH transgenic mice immunized with fHbp vaccines.

FIG. 14. Binding of fH with a K241E mutant of fHbp ID 1 and its bindingto MAb JAR 5 are shown in panels A and B, respectively. Binding of fHwith an E241K mutant of fHbp ID 15 and its binding to MAb JAR 5 areshown in panels C and D, respectively. fH or anti-fHbp MAb binding tofHbp was measured as described in Example 2.

FIG. 15. Binding of fH or anti-fHbp MAbs to H119A and R130A singlemutants of fHbp ID 1, as measured by ELISA. Binding of human fH (panelA), and anti-fHbp MAbs JAR 5 (panel B), or JAR 4 (panel C), was measuredas described in Example 2.

FIG. 16. Schematic representation of the six most common fHbp modulargroups, designated I to VI. The variable segments are each derived fromone of two genetic lineages, designated α (shown in gray) or β (white).The α and β lineages can also be designated as lineages 1 and 2,respectively, according to the nomenclature adopted by thepubmlst.org/neisseria/fHbp/ website. Segment V_(A) began at amino acidresidue 8 and extended to position 73 while segment V_(B) began atposition 79 and extended to position 93 (numbering of the amino acidresidue based on the sequence of fHbp ID 1). Segment V_(C) began atamino acid residue 98 and extended to position 159 while segment V_(D)began at position 161 and extended to position 180. Segment V_(E) beganat amino acid residue 186 and extended to position 253. Of the 70 fHbpamino acid sequence variants analyzed, 33 contained only α typesegments, and 7 contained only β type segments, which were designated asmodular groups I and II, respectively. The remaining 30 fHbp variantswere natural chimeras with different combinations of α and β segmentsand could be assigned to one of four modular groups (III-VI). Therelationship between the modular group and Masignani variant groupdesignation, and the number of unique sequences observed within eachfHbp modular group, are shown. The modular architecture of theengineered (non-naturally occurring) fHbp chimera I is shown as the lastmodular schematic in FIG. 16. For a chimeric protein engineered fromfHbp ID 1 and ID 77 “chimera I” (Beernink et al. (2008) Infec. Immun.76:2568-2575), four amino acid residues, GEHT (SEQ ID NO:27) at position136 to 139 represents the junction point in the V_(C) segment (See FIG.19). ID refers to fHbp sequence peptide identification number asdescribed on the public website, (hypertext transferprotocol)://pubmlst.org/neisseria/fHbp/.

FIG. 17. Binding of fH with recombinant fHbp mutant S41P (mutant of fHbpID 15). Binding of fH with an S41P mutant of fHbp ID 15 is shown inPanel A. Binding of the S41P mutant of fHbp ID15 to MAb JAR 5 and to MAbJAR31 are shown in panels B and C, respectively. “Pep28” is fHbp ID 28;“Pepl” is fHbp ID 1; “Pep 15 WT” is fHbp ID 15; and “Pep 15 S41P” is theS41P mutant of fHbp ID 15.

FIG. 18. Binding of human fH to R41S mutant of the fHbp chimera I(Beernink et al. (2008) Infec. Immun. 76:2568-2575) (panel A) andcorresponding binding of JAR 5 (Panel B).

FIG. 19A. Alignment of fHbp sequences of natural variants and a man-madechimera (chimera I; Beernink et al. (2008) Infec. Immun. 76:2568-2575).fHbp ID 1 is in modular group I (all five variable segments, A-E, arederived from α lineages as defined by Beernink and Granoff (2009)Microbiology 155:2873-83). fHbp ID 28 is in modular group II (all fivesegments are derived from β lineages). fHbp ID 15 is a natural chimera(modular group IV with a β A segment and α B, C, D and E segments). Theβ-type A segment (V_(A); residues 8-73) of fHbp ID 28 is shown forcomparison with the corresponding A segment (V_(A)) of fHbp ID 15, whichalso has a β-type A segment (V_(A)β). The residues changed in theE218A/E239A double mutant fHbp are shown in rectangles. FIG. 19B,Alignment of the A segment (amino acid residues 8 to 73) of fHbp ID 1and fHbp ID 77. FIG. 19C, Alignment of the C segment (amino acidresidues 98-159) of fHbp ID 1 and fHbp ID 77. The junction point is atresidue 136. Chimeric fHbp includes the amino acid sequences from ID1 upto residue G136, and the sequence of fHbp ID 77 from residue 136 to theC terminus. FIG. 19D, Alignment showing natural polymorphisms at aminoacid position 41 (number according to that of fHbp ID 1); some variantshave arginine (R41, ID 1, 19, 4, 9 and 74) while other variants haveserine (S41, ID 55, 15) or proline (P41, ID 28). ID refers to fHbpsequence ID; MG refers to fHbp modular group; and VG refers to variantgroup. FIG. 19E, Alignment of fHbp ID 1, fHbp ID 77 and chimera I.Shaded residues in fHbp ID 77 highlight the residues in segment V_(C)that are different from the corresponding positions in chimera I. Boldedand shaded residues correspond to K113, K119, and D121, in order ofN-terminus to C-terminus.

FIG. 20. Binding of fH or an anti-fHbp MAb to K113A, K119A, and D121Asingle mutants of fHbp ID 77, as measured by ELISA. Binding of human fH(panel A), and anti-fHbp MAb JAR 31 (panel B) was measured as describedin Example 2.

FIG. 21. Binding of fH or an anti-fHbp MAb to R41S/K113A, R41S/K119A,and R41S/D121A double mutants of fHbp ID 77, as measured by ELISA.Binding of human fH (panel A), and anti-fHbp MAb JAR 31 (panel B) wasmeasured as described in Example 2.

FIG. 22. Binding of fH or an anti-fHbp MAb to K113A/D121A double mutantand R41S/K113A/D121A triple mutant of fHbp ID 77, as measured by ELISA.Binding of human fH (panel A), anti-fHbp MAb JAR 4 (panel B), andanti-fHbp MAb JAR 31 (panel C) was measured as described in Example 2.

FIG. 23. Binding of fH to mutants of fHbp ID 22, as measured by ELISA.Binding of human fH to D211A, R80A, or wild-type fHbp (panel A), toE218A, E248A, or wild-type fHbp (panel B), and to R41S, Q38A, Q126A, orwild-type fHbp (panel C) was measured as described in Example 2.

FIG. 24. Binding of anti-fHbp MAb JAR31 to mutants of fHbp ID 22, asmeasured by ELISA. Binding of R80A and D211A mutants (panel A), andbinding of E218A and E248A mutants (panel B) to JAR31 was measured asdescribed in Example 2.

FIG. 25. Binding of anti-fHbp MAb JAR4 to mutants of fHbp ID 22, asmeasured by ELISA. Binding of R80A and D211A mutants (panel A), andbinding of E218A and E248A mutants (panel B) to JAR 4 was measured asdescribed in Example 2.

FIG. 26. Binding of anti-fHbp MAb JAR35 to mutants of fHbp ID 22, asmeasured by ELISA. Binding of R80A and D211A mutants (panel A), andbinding of E218A and E248A mutants (panel B) to JAR35 was measured asdescribed in Example 2.

FIG. 27. Binding of fH or anti-fHbp MAbs to a T221A/H223A double mutant,or a G236I mutant, of fHbp ID 22, as measured by ELISA. Binding of humanfH (panel A), and anti-fHbp MAbs JAR 31 (panel B), JAR 35 (panel C), orJAR 4 (panel D), was measured as described in Example 2.

FIG. 28. Binding of fH or anti-fHbp MAbs to R41S, Q38A, and A235Gmutants of fHbp ID 22, as measured by ELISA. Binding of human fH (panelA), and anti-fHbp MAbs JAR 31 (panel B), or JAR 35 (panel C), wasmeasured as described in Example 2.

FIG. 29. Binding of fH or an anti-fHbp MAb to Q126A, D201A, and E202Amutants of fHbp ID 22, as measured by ELISA. Binding of human fH (panelA), and anti-fHbp MAb JAR 35 (panel B) was measured as described inExample 2.

FIG. 30. Binding to mutants of fHbp ID 28 (variant group 3). Panels Aand C. Binding of fH to K199A, E217A, and E218A mutants as measured byELISA. Panels B and D. Binding of anti-fHbp MAb JAR 31 (panel B), andanti-fHbp MAb JAR 33 (panel D, fHbp wildtype ID 28 WT and E218A mutantonly) are shown.

FIG. 31 depicts serum bactericidal titers of wildtype BALB/c miceimmunized with the indicated mutants of fHbp ID 1 vaccine and measuredagainst group B strain H44/76 (fHbp ID 1).

FIG. 32 depicts serum bactericidal titers of mice immunized with theindicated mutants of fHbp ID 22 as measured against group W-135 strainGhana 7/04 (fHbp ID 23). Upper panel, mutant vaccines with titers thatwere not significantly different from that of the wildtype (WT) fHbp ID22 vaccine (P>0.10). Lower panel, mutant vaccines that elicitedsignificantly lower titers than the control WT ID 22 vaccine (P<0.05).

FIG. 33 depicts bactericidal titers of mice immunized with a tripleR41S/K113A/D121A mutant of fHbp ID 77 as measured against group W-135strain Ghana 7/04 (fHbp ID 23).

FIG. 34. Alignment of fHbp ID 1 (SEQ ID NO:1), fHbp ID 22 (SEQ ID NO:2),fHbp ID 77 (SEQ ID NO:4), fHbp ID 28 (SEQ ID NO:3), and ID1/ID77 chimera(SEQ ID NO:8) amino acid sequences. ID 28 is shown as a referencesequence for fHbp variant group 3. Predicted factor H binding interfaceresidues with hydrogen bond or ionic interactions (highlighted in gray)from a crystal structure of fHbp ID1 in a complex with a fragment of fH,as described in Schneider et al. ((2009) Nature 458:890-3). GEHT (SEQ IDNO:27) (in bold) at position 136 to 139 represents the junction pointbetween ID 1 and ID 77 for the chimeric fHbp.

FIG. 35. Alignment of fHbp ID 1 (SEQ ID NO:1), fHbp ID 22 (SEQ ID NO:2),fHbp ID 77 (SEQ ID NO:4), fHbp ID 28 (SEQ ID NO:3), and ID1/ID77 chimera(SEQ ID NO:8) amino acid sequences. Residues highlighted in grayindicate residues mutated and summarized in Table 7.

FIG. 36 depicts a model of fHbp in a complex with a fragment of fH. Thepositions of the amino acid residues known to affect the epitopes ofanti-fHbp mAb JAR 3 and JAR 5 (G121 and K122) and mAb 502 (R204) aredepicted.

FIGS. 37A-37D depict binding of human IgGa mouse chimeric fHbp-specificmAbs to fHbp as measured by ELISA (FIG. 37A), plasmon resonance (FIG.37B) or to live bacteria by flow cytometry (FIG. 37C, mAbs alone; andFIG. 37D, mAbs in the presence of 20% human serum depleted of IgG).

FIGS. 38A-38C. FIGS. 38A-38B depict C1q-dependent C4b deposition fromcomplement activation on encapsulated group B bacteria of strain H44/76by human IgG1 mouse chimeric anti-fHbp mAbs JAR 3, JAR 5 and mAb 502.FIG. 38A, C1q-depleted human serum as complement source; FIG. 38B,C1q-depleted serum that had been repleted with purified C1q proteinprior to the reactions. FIG. 38C depicts human complement-mediatedbactericidal activity of the respective mAbs as measured against group Bstrain H44/76.

FIGS. 39A-39C depict inhibition of binding of fH by anti-fHbp mAbs asmeasured by ELISA with fHbp adhered to the wells of the microtiter plate(FIG. 39A), and with live bacteria of group B strain H44/76 as measuredby flow cytometry (FIGS. 39B and 39C).

FIGS. 40A-40C depict binding of fH to mutants of group B H44/76 withgenetic inactivation of fHbp expression, or both fHbp and NspA. FIG.40A, binding of a control anti-PorA mAb; FIGS. 40B and 40C, binding offH in human serum depleted of IgG.

FIGS. 41A-41E depict bactericidal activity of human IgG mouse chimericanti-fHbp mAbs measured against a mutant of group B H44/76 with geneticinactivation of NspA. FIGS. 41A, 41B, and 41C: anti-fHbp mAbs JAR 3, JAR5 and mAb 502, respectively; FIGS. 41D and 41E: control anti-PorA andanti-capsular mAbs, respectively.

FIG. 42 depicts bactericidal activity against a capsular group A strain(Senegal 1/99) of an anti-NspA antibody against a fHbp knockout of agroup A strain (top panel) or control anti-PorA mAb P1.9 (lower panel).

FIG. 43, Panels A-C, depicts serum anti-fHbp antibody responses ofwildtype mice immunized with recombinant fHbp vaccine or native outermembrane vesicle vaccines from mutants of group B strain H44/76 withover-expressed fHbp or fHbp knockout. Anti-fHbp antibody responses tovaccination as measured by ELISA (Panel A), or the ability of serumanti-fHbp antibodies to inhibit binding of fH to fHbp (Panels B and C,also by ELISA). Mice were immunized with recombinant fHbp ID 1 vaccine(filled triangles), or NOMV vaccines prepared from mutants of group Bstrain H44/76 with over-expressed of fHbp ID 1 (open circles) or a fHbpknock-out (filled circles).

FIG. 44 presents an amino acid sequence of a Neisserial surface proteinA (NspA) polypeptide (SEQ ID NO:15).

FIG. 45. Amino acid sequences of various naturally-occurring factor Hbinding proteins (fHbps): fHbp ID 1, fHbp ID 15, fHbp ID 22, fHbp ID 28,fHbp ID 77, and chimera I (Beernink et al. (2008) Infec. Immun.76:2568-2575). FHbp ID sequences are shown without a leader sequence. Inthe sequence shown for chimera I, the lower case letters correspond tothe amino acid sequence that is derived from fHbp ID 1 while the uppercase letters correspond to the amino acid that is derived from ID 77.

Before the present invention and specific exemplary embodiments of theinvention are described, it is to be understood that this invention isnot limited to particular embodiments described, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either both ofthose included limits are also included in the invention.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to amino acid modifications, including amino acidsubstitutions, relative to a reference amino acid sequence arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination were individually and explicitlydisclosed, to the extent that such combinations embrace polypeptideshaving desired features, e.g., non-naturally occurring fHbp polypeptideshaving a lower affinity for a human fH than that of fHbp ID 1. Inaddition, all sub-combinations of such amino acid modifications(including amino acid substitutions) listed in the embodimentsdescribing such amino acid modifications are also specifically embracedby the present invention and are disclosed herein just as if each andevery such sub-combination of such amino acid modifications wasindividually and explicitly disclosed herein.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anantigen” includes a plurality of such antigens and reference to “theprotein” includes reference to one or more proteins, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

Factor H binding proteins that can elicit antibodies that arebactericidal for at least one strain of N. meningitidis, and methods ofuse such proteins, are provided.

Definitions

“Factor H Binding Protein” (fHbp), which is also known in the literatureas GNA1870, GNA 1870, ORF2086, LP2086 (lipoprotein 2086), and “741”refers to a class of N. meningitidis polypeptides. It is found in natureas a lipoprotein on the surface of the bacterium. N. meningitidisstrains. fHbps have been sub-divided into three fHbp variant groups(referred to as variant 1 (v.1), variant 2 (v.2), and variant 3 (v.3) insome reports (Masignani et al. (2003) J Exp Med 197:789-99) and Family Aand B in other reports (see, e.g., Fletcher et al. (2004) Infect Immun72:2088-2100)) based on amino acid sequence variability and immunologiccross-reactivity (Masignani et al. (2003) J Exp Med 197:789-99). Eachunique fHbp found in N. meningitidis is also assigned a fHbp peptide IDaccording to neisseria.org or pubmlst.org/neisseria/fHbp/ website.Because the length of variant 2 (v.2) fHbp protein (from strain 8047,fHbp ID 77) and variant 3 (v.3) fHBP (from strain M1239, fHbp ID 28)differ by −1 and +7 amino acid residues, respectively, from that of MC58(fHbp ID 1), the numbering used to refer to residues for v.2 and v.3fHbp proteins differs from numbering based on the actual amino acidsequences of these proteins. Thus, for example, reference to a leucineresidue (L) at position 166 of the v.2 or v.3 fHBP sequence refers tothe residue at position 165 of the v.2 protein and at position 173 inthe v.3 protein.

Human factor H (“human fH”) as used herein, refers to a proteincomprising an amino acid sequence as shown in FIG. 9B (SEQ ID NO:9), andnaturally-occurring human allelic variants thereof.

The term “heterologous” or “chimeric” refers to two components that aredefined by structures derived from different sources or progenitorsequences. For example, where “heterologous” is used in the context of achimeric polypeptide, the chimeric polypeptide can include operablylinked amino acid sequences that can be derived from differentpolypeptides of different phylogenic groupings (e.g., a first componentfrom an α and a second component from a β progenitor amino acidsequences). A chimeric polypeptide containing two or more definedsegments, each of which is from a different progenitor, can benaturally-occurring or man-made (non-naturally-occurring). See BeerninkP T, Granoff D M (2009) Microbiology 155:2873-83 for more detail onnaturally-occurring chimeras. Non-naturally occurring chimeras refers to“man-made chimeras” and encompass fHbp with heterologous components thatare not found in nature.

A “heterologous” or “chimeric” polypeptide may also contain two or moredifferent components, each derived from a different fHbp (e.g. variant1, 2, or 3). The component may be operably linked at any position alongthe length of the fHbp polypeptide.

“Heterologous” in the context of a polynucleotide encoding any chimericpolypeptide as described above can include operably linked nucleic acidsequence that can be derived from different genes (e.g., a firstcomponent from a nucleic acid encoding a fHBP v.1 polypeptide and asecond component from a nucleic acid encoding a fHBP v.2 polypeptide) ordifferent progenitor amino acid sequences (α or β).

Other exemplary “heterologous” nucleic acids include expressionconstructs in which a nucleic acid comprising a coding sequence isoperably linked to a regulatory element (e.g., a promoter) that is froma genetic origin different from that of the coding sequence (e.g., toprovide for expression in a host cell of interest, which may be ofdifferent genetic origin relative to the promoter, the coding sequenceor both). For example, a T7 promoter operably linked to a polynucleotideencoding an fHbp polypeptide or domain thereof is said to be aheterologous nucleic acid.

“Heterologous” in the context of recombinant cells can refer to thepresence of a nucleic acid (or gene product, such as a polypeptide) thatis of a different genetic origin than the host cell in which it ispresent. For example, a Neisserial amino acid or nucleic acid sequenceof one strain is heterologous to a Neisserial host of another strain.

“Derived from” in the context of an amino acid sequence orpolynucleotide sequence (e.g., an amino acid sequence “derived from”fHbp ID 1) is meant to indicate that the polypeptide or nucleic acid hasa sequence that is based on that of a reference polypeptide or nucleicacid (e.g., a naturally occurring fHbp protein or encoding nucleicacid), and is not meant to be limiting as to the source or method inwhich the protein or nucleic acid is made. Non-limiting examples ofreference polypeptides and reference polynucleotides from which an aminoacid sequence or polynucleotide sequence may be “derived from” include anaturally-occurring fHbp, fHbp ID1, and a non-naturally-occurring fHbp.“Derived from” in the context of bacterial strains is meant to indicatethat a strain was obtained through passage in vivo, or in in vitroculture, of a parental strain and/or is a recombinant cell obtained bymodification of a parental strain.

“Conservative amino acid substitution” refers to a substitution of oneamino acid residue for another sharing chemical and physical propertiesof the amino acid side chain (e.g., charge, size,hydrophobicity/hydrophilicity). “Conservative substitutions” areintended to include substitution within the following groups of aminoacid residues: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr;lys, arg; and phe, tyr. Guidance for such substitutions can be drawnfrom alignments of amino acid sequences of polypeptides presenting theepitope of interest.

The term “protective immunity” means that a vaccine or immunizationschedule that is administered to a mammal induces an immune responsethat prevents, retards the development of, or reduces the severity of adisease that is caused by Neisseria meningitidis, or diminishes oraltogether eliminates the symptoms of the disease. Protective immunitycan be accompanied by production of bactericidal antibodies. It shouldbe noted that production of bactericidal antibodies against Neisseriameningitidis is accepted in the field as predictive of a vaccine'sprotective effect in humans. (Goldschneider et al. (1969) J. Exp. Med.129:1307; Borrow et al. (2001) Infect Immun. 69:1568).

The phrase “a disease caused by a strain of Neisseria meningitidis”encompasses any clinical symptom or combination of clinical symptomsthat are present in an infection of a human with a Neisseriameningitidis. These symptoms include but are not limited to:colonization of the upper respiratory tract (e.g. mucosa of thenasopharynx and tonsils) by a pathogenic strain of Neisseriameningitidis, penetration of the bacteria into the mucosa and thesubmucosal vascular bed, septicemia, septic shock, inflammation,haemorrhagic skin lesions, activation of fibrinolysis and of bloodcoagulation, organ dysfunction such as kidney, lung, and cardiacfailure, adrenal hemorrhaging and muscular infarction, capillaryleakage, edema, peripheral limb ischaemia, respiratory distresssyndrome, pericarditis and meningitis.

The phrase “specifically binds to an antibody” or “specificallyimmunoreactive with”, in the context of an antigen (e.g., a polypeptideantigen) refers to a binding reaction which is based on and/or isprobative of the presence of the antigen in a sample which may alsoinclude a heterogeneous population of other molecules. Thus, underdesignated conditions, the specified antibody or antibodies bind(s) to aparticular antigen or antigens in a sample and do not bind in asignificant amount to other molecules present in the sample.“Specifically binds to an antibody” or “specifically immunoreactivewith” in the context of an epitope of an antigen (e.g., an epitope of apolypeptide) refers to a binding reaction which is based on and/or isprobative of the presence of the epitope in an antigen (e.g.,polypeptide) which may also include a heterogeneous population of otherepitopes, as well as a heterogeneous population of antigens. Thus, underdesignated conditions, the specified antibody or antibodies bind(s) to aparticular epitope of an antigen and do not bind in a significant amountto other epitopes present in the antigen and/or in the sample.

The phrase “in a sufficient amount to elicit an immune response” meansthat there is a detectable difference between an immune responseindicator measured before and after administration of a particularantigen preparation Immune response indicators include but are notlimited to: antibody titer or specificity, as detected by an assay suchas enzyme-linked immunoassay (ELISA), bactericidal assay, flowcytometry, immunoprecipitation, Ouchterlony immunodiffusion; bindingdetection assays of, for example, spot, Western blot or antigen arrays;cytotoxicity assays, etc.

A “surface antigen” is an antigen that is present in a surface structureof Neisseria meningitidis (e.g. the outer membrane, capsule, pili,etc.).

“Isolated” refers to an entity of interest that is in an environmentdifferent from that in which the compound may naturally occur.“Isolated” is meant to include compounds that are within samples thatare substantially enriched for the compound of interest and/or in whichthe compound of interest is partially or substantially purified.

“Enriched” means that a sample is non-naturally manipulated (e.g., by anexperimentalist or a clinician) so that a compound of interest ispresent in a greater concentration (e.g., at least a three-fold greater,at least 4-fold greater, at least 8-fold greater, at least 64-foldgreater, or more) than the concentration of the compound in the startingsample, such as a biological sample (e.g., a sample in which thecompound naturally occurs or in which it is present afteradministration), or in which the compound was made (e.g., as in abacterial polypeptide, antibody, polypeptide, and the like)

A “knock-out” or “knockout” in the context of a target gene refers to analteration in the sequence of the gene that results in a decrease offunction of the target gene, e.g., such that target gene expression isundetectable or insignificant, and/or the gene product is not functionalor not significantly functional. For example, a “knockout” of a geneinvolved in LPS synthesis indicates means that function of the gene hasbeen substantially decreased so that the expression of the gene is notdetectable or only present at insignificant levels and/or a biologicalactivity of the gene product (e.g., an enzymatic activity) issignificantly reduced relative to prior to the modification or is notdetectable. “Knock-outs” encompass conditional knock-outs, wherealteration of the target gene can occur upon, for example, exposure to apredefined set of conditions (e.g., temperature, osmolarity, exposure tosubstance that promotes target gene alteration, and the like. A“knock-in” or “knockin” of a target gene refers to a genetic alterationin a gene that that results in an increase in a function provided by thetarget gene.

fHbp Polypeptides with Altered FH Binding Properties

Before describing further fHbps contemplated by the present disclosure,it is helpful to describe some naturally-occurring fHbps. Uniquenaturally-occurring fHbps found in N. meningitidis are each assigned afHbp peptide ID according to neisseria.org andpubmlst.org/neisseria/fHbp websites. This convention of naming fHbpswill be adopted throughout the present disclosure.

For convenience and clarity, the native amino acid sequence of fHbp ID 1(v.1 fHbp of the N. meningitidis strain MC58) is selected as a referencesequence for all naturally occurring and non-naturally occurring fHbpamino acid sequences, encompassing chimeric and/or variants of fHbpsdescribed herein. The amino acid sequence of fHbp ID 1 is shown in FIG.45 and presented below:

fHbp ID1 (SEQ ID NO: 1)CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTAFQTEQIQDSEHSGKMVAKRQFRIGDIAGEHTSFDKLPEGGRATYRGTAFGSDDAGGKLTYTIDFAAKQGNGKIEHLKSPELNVDLAAADIKPDGKRHAVISGSVLYNQAEKGSYSLGIFGGKAQEVAGSAEVKTVNGIRHIG LAAKQ.

In referring to an amino acid residue position in a fHbp, the positionnumber used herein corresponds to the amino acid residue number of fHbpID 1. See FIG. 19 for an alignment of various fHbps and the amino acidresidues in each fHbp corresponding to those of fHbp ID 1. As seen inFIG. 19 and SEQ ID NO: 1, position number 1 refers to the first aminoacid residue shown in fHbp ID 1, which is a cysteine. The fHbp referredto herein may sometimes contain an additional leader sequence at theN-terminus. For example, fHbp ID 1 may have a leader sequence ofMNRTAFCCLSLTTALILTA (SEQ ID NO:16) at the N-terminus. However, aminoacid position number 1 in any fHbp is still defined herein as theposition that corresponds to the cysteine at amino acid position 1 shownabove for fHbp ID 1 in an alignment, which amino acid is the firstresidue after the leader sequence, if present. See FIG. 19 for details.

The present disclosure provides fHbps, compositions comprising same, andmethods of use of the fHbps and compositions. A subject fHbp has a loweraffinity for human fH than a corresponding reference fHbp (e.g. a fHbpthat is naturally-occurring; or other reference fHbp). Because ahigh-affinity fHbp has a high probability to be complexed with fH, thebound fH can mask one or more epitopes on the fHbp from a host's immunesystem. Accordingly, fHbp that is complexed and/or bound with fH may notbe as effective an immunogen as an fHbp that is not so complexed.Conversely, fHbps that have a relatively low affinity for fH, whenadministered as an immunogen (e.g. in a vaccine composition), canpresent epitopes to the immune system of an immunized host that an fHbpthat has high affinity for fH does not, as such epitopes may be maskedby bound fH. The subject fHbps have a low affinity for human fH and areuseful in eliciting bactericidal antibodies and/or providing protectiveimmunity against N. meningitidis. A subject fHbp is a non-naturallyoccurring fHbp. A non-naturally occurring fHbp is not found in natureand is made by a human and/or intentionally modified by a human Anon-naturally occurring subject fHbp can be made via chemical synthesisor recombinant methods.

As used herein, “low affinity”, “lower affinity”, or “low fH binder”refers to fHbps that have a binding affinity for a human fH that is aslow as or lower than that of fHbp ID 1. Accordingly, subject fHbps canencompass fHbp ID 14 and fHbp 15 since fHbp ID 14 and fHbp ID 15 have alower affinity for human fH relative to fHbp ID 1.

The binding affinity of low-affinity fHbps and human fH can be no morethan about 100%, no more than about 95%, no more than about 90%, no morethan about 85%, more than about 80%, no more than about 75%, no morethan about 70%, no more than about 65% fold, no more than about 60%, nomore than about 50%, no more than about 45% or less of the affinity ofhigh-affinity fHbp (e.g. fHbp ID 1) and human fH. For example, a subjectfHbp can have an affinity for human fH that is less than about 50% ofthe affinity of fHbp ID 1 for human fH.

In some embodiments, the binding affinity of a subject non-naturallyoccurring fHbp for human fH is 85% or less of the binding affinity of awildtype fHbp for human fH. For example, in some embodiments, thebinding affinity of a subject non-naturally occurring fHbp for human fHis from about 85% to about 75%, from about 75% to about 65%, from about65% to about 55%, from about 55% to about 45%, from about 45% to about35%, from about 35% to about 25%, from about 25% to about 15%, fromabout 15% to about 10%, from about 10% to about 5%, from about 5% toabout 2%, from about 2% to about 1%, or from about 1% to about 0.1%, orless than 0.1%, of the binding affinity of a wildtype fHbp for human fH.As an example, in some embodiments, the binding affinity of a subjectnon-naturally occurring fHbp for human fH is from about 85% to about75%, from about 75% to about 65%, from about 65% to about 55%, fromabout 55% to about 45%, from about 45% to about 35%, from about 35% toabout 25%, from about 25% to about 15%, from about 15% to about 10%,from about 10% to about 5%, from about 5% to about 2%, from about 2% toabout 1%, or from about 1% to about 0.1%, or less than 0.1%, of thebinding affinity of fHbp ID 1 for human fH.

For example, in some embodiments, the binding affinity of a subjectmutant of fHbp ID1 (e.g., an R41S, R41A, R130A, H119A, E218A, or a E239Amutant of fHbp ID1) for human fH is from about 85% to about 75%, fromabout 75% to about 65%, from about 65% to about 55%, from about 55% toabout 45%, from about 45% to about 35%, from about 35% to about 25%,from about 25% to about 15%, from about 15% to about 10%, from about 10%to about 5%, from about 5% to about 2%, from about 2% to about 1%, orfrom about 1% to about 0.1%, or less than 0.1%, of the binding affinityof fHbp ID1 for human fH.

As another example, in some embodiments, the binding affinity of asubject mutant of fHbp ID 4, ID 9, or ID 74 (e.g., an R41S mutant offHbp ID4, ID9, or ID74) for human fH is from about 85% to about 75%,from about 75% to about 65%, from about 65% to about 55%, from about 55%to about 45%, from about 45% to about 35%, from about 35% to about 25%,from about 25% to about 15%, from about 15% to about 10%, from about 10%to about 5%, from about 5% to about 2%, from about 2% to about 1%, orfrom about 1% to about 0.1%, or less than 0.1%, of the binding affinityof fHbp ID4, ID9, or ID74 for human fH, or of the binding affinity offHbp ID1 for human fH.

As another example, in some embodiments, the binding affinity of asubject mutant of fHbp ID 22 (e.g., an R80A, D211A, E218A, E248A, G236I,or T221A/H223A mutant of fHbp ID22) for human fH is from about 85% toabout 75%, from about 75% to about 65%, from about 65% to about 55%,from about 55% to about 45%, from about 45% to about 35%, from about 35%to about 25%, from about 25% to about 15%, from about 15% to about 10%,from about 10% to about 5%, from about 5% to about 2%, from about 2% toabout 1%, or from about 1% to about 0.1%, or less than 0.1%, of thebinding affinity of fHbp ID 22 for human fH, or of the binding affinityof fHbp ID1 for human fH.

As another example, in some embodiments, the binding affinity of asubject mutant of fHbp ID 77 (e.g., an R41S/K113A, R41S/K119A,R41S/D121A, or a R41S/K113A/D121A mutant of fHbp ID 77) for human fH isfrom about 85% to about 75%, from about 75% to about 65%, from about 65%to about 55%, from about 55% to about 45%, from about 45% to about 35%,from about 35% to about 25%, from about 25% to about 15%, from about 15%to about 10%, from about 10% to about 5%, from about 5% to about 2%,from about 2% to about 1%, or from about 1% to about 0.1%, or less than0.1%, of the binding affinity of fHbp ID 77 for human fH, or of thebinding affinity of fHbp ID1 for human fH.

As another example, in some embodiments, the binding affinity of asubject mutant of fHbp ID 28 (e.g., an E218A mutant or fHbp ID 28; aK199A mutant of fHbp ID 28) for human fH is from about 85% to about 75%,from about 75% to about 65%, from about 65% to about 55%, from about 55%to about 45%, from about 45% to about 35%, from about 35% to about 25%,from about 25% to about 15%, from about 15% to about 10%, from about 10%to about 5%, from about 5% to about 2%, from about 2% to about 1%, orfrom about 1% to about 0.1%, or less than 0.1%, of the binding affinityof fHbp ID 28 for human fH, or of the binding affinity of fHbp ID1 forhuman fH.

Binding affinity can be described in terms of the dissociation constant(K_(D)). Low-affinity fHbps and human fH can have a dissociationconstant (K_(D); M) that is at least more than about 80%, at least morethan about 100%, at least more than about 120%, at least more than about140%, at least more than about 160%, at least more than about 200%, ormore than K_(D) of high affinity fHbps (e.g. fHbp ID 1) and human fH.The K_(D) of a low-affinity fHbp can also be described as about 2× (2times), about 3×, about 5×, about 10×, about 15×, about 20×, up to about50 or more times the K_(D) of fHbp ID 1. For example, a subject fHbp andhuman fH can have a K_(D) that is 110% of or about 15× that of fHbp ID 1and human fH.

As used herein, “lower affinity for human fH than a corresponding fHbp”is used to describe fHbps that have a binding affinity lower than acorresponding reference fHbp.

In many cases, the corresponding fHbp (the “reference fHbp”) used tocompare the binding affinities of subject fHbps is fHbp ID 1. Othercorresponding fHbp that can be representative as a reference includevariant 2 fHbp (e.g. fHbp ID 22 or 77), variant 3 (e.g. fHbp ID 28)(Masignani et al (2003) J Exp Med 197:789-99 and Pajon R et al (2010)Vaccine 28:2122-9), other variant 1 fHbps (e.g. fHbp ID 4, 9, or 94), anaturally-occurring chimeric, or a man-made chimeric fHbp.

The amino acid sequences of some examples of naturally-occurring fHbpsand a man-made chimeric are provided below and shown in FIG. 45.

FHbp ID 22 (SEQ ID NO: 2)CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPSGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTPEQNVELASAELKADEKSHAVILGDTRYGGEEKGTYHLALFGDRAQEIAGSATVKIREKVHEIGI AGKQ FHbp ID 28(SEQ ID NO: 3) CSSGGGGSGGGGVAADIGTGLADALTAPLDHKDKGLKSLTLEDSIPQNGTLTLSAQGAEKTFKAGDKDNSLNTGKLKNDKISRFDFVQKIEVDGQTITLASGEFQIYKQNHSAVVALQIEKINNPDKTDSLINQRSFLVSGLGGEHTAFNQLPGGKAEYHGKAFSSDDPNGRLHYSIDFTKKQGYGRIEHLKTLEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIG EKVHEIGIAGKQFHbp ID 77 (SEQ ID NO: 4)CSSGGGGVAADIGARLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGI AGKQ FHbp ID 15(SEQ ID NO: 5) CSSGGGGSGGGGVAADIGAGLADALTAPLDHKDKGLKSLTLEDSISQNGTLTLSAQGAERTFKAGDKDNSLNTGKLKNDKISRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSGKMVAKRQFRIGDIVGEHTSFGKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVDLAAADIKPDEKHHAVISGSVLYNQAEKGSYSLGIFGGQAQEVAGSAEVET ANGIRHIGLAAKQFHbp ID 6 (SEQ ID NO: 6)CSSGGGGVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVNGQLITLESGEFQVYKQSHSALTALQTEQVQDSEHSRKMVAKRQFRIGDIAGEHTSFDKLPKGDSATYRGTAFGSDDAGGKLTYTIDFAAKQGYGKIEHLKSPELNVDLAAAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVKTANGIRHIG LAAKQ FHbp ID 14(SEQ ID NO: 7) CSSGGGGVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQVYKQSHSALTALQTEQEQDPEHSGKMVAKRRFKIGDIAGEHTSFDKLPKDVMATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEKHHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVETANGIHHIG LAAKQ Chimera I(SEQ ID NO: 8) cssggggvaadigagladaltapldhkdkglqsltldqsvrkneklklaaqgaektygngdslntgklkndkvsrfdfirqievdgqlitlesgefqvykqshsaltafqteqiqdsehsgkmvakrqfrigdiaGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGI AGKQ(Beernink et al. (2008) Infec. Immun. 76:2568-2575). As noted in FIG.45, the lower case letters correspond to the amino acid sequence that isderived from fHbp ID 1 while the upper case letters correspond to theamino acid that is derived from fHbp ID 77. Position corresponding toR41 in fHbp ID 1 is the bolded lower case “r”.

The corresponding fHbp can be a naturally-occurring and/or non-naturallyoccurring (e.g. man-made chimeric) fHbp from which the subject fHbp isderived. Naturally-occurring chimeric encompass fHbp that have variablesegments derived from different progenitors (α or β). Due to thevariable segments, the molecular architecture has been shown to bemodular and fHbp variants can be subclassified in modular groupsaccording to different combinations of five variable segments, eachderived from one of two genetic lineages, designated α- or β-types(Pajon R et al. (2010) Vaccine 28:2122-9; Beernink P T, Granoff D M(2009) Microbiology 155:2873-83). Six modular groups, designated I to VIaccount for >95% of all known fHbp variants (Pajon R et al. (2010)Vaccine 28:2122-9). See FIG. 16 for modular group architectures ofnaturally-occurring fHbps.

The corresponding fHbp can be a fHbp that has a high amino acid sequenceidentity as the subject fHbp (e.g. at least about 99%, at least about95%, at least about 90%, at least about 85%, at least about 80%, or atleast about 75% amino acid sequence identity) either in a segment (e.g.variable segment as defined in a modular architecture) or in thefull-length mature protein.

Corresponding fHbps used as references to compare the binding affinitiesof subject fHbp can also encompass fHbps that have one or more segmentsof the same progenitor (α or β) in corresponding segments of the subjectfHbp.

The subject fHbp can comprise an amino acid sequence having at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, amino acidsequence identity with a reference fHbp; and differs from the amino acidsequence of the reference fHbp by from 1 amino acid (aa) to 10 aminoacids, e.g., differs from the amino acid sequence of the reference fHbpby 1 aa, 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, or 10 aa. Thus,e.g., a subject fHbp can have at most one, at most two, at most three,at most four, up to at most 10 or more modifications (e.g.substitutions, deletions, or insertions) relative to a naturallyoccurring and/or non-naturally-occurring (e.g. chimeric) fHbp from whichthe subject fHbp is derived. The one or more amino acid alterations candecrease the affinity of the fHbp for human fH relative to a fHbp thatis not altered. As noted above, fHbps from which the subject fHbp arederived encompass naturally occurring fHbps and non-naturally occurringfHbp. Non-naturally occurring fHbps can encompass man-made chimeras,such as those known in the art and described in PCT application numberWO 2009/114485, disclosure of which is incorporated herein by reference.

Thus, in some embodiments, a subject fHbp comprises a single amino acidsubstitution relative to a reference fHbp (e.g., where the referencefHbp is a naturally-occurring fHbp (e.g. fHbp ID 1, or a man-madechimeric). In some embodiments, a subject fHbp comprises a single aminoacid substitution (i.e., only one amino acid substitution) relative to anaturally-occurring fHbp (e.g., fHbp ID 6, fHbp ID 14, fHbp ID 15, fHbpID 22, fHbp ID 28, fHbp ID 77, or another naturally-occurring fHbp). Theamino acid sequences of fHbp ID 1, fHbp ID 15, fHbp ID 22, fHbp ID 28,and fHbp ID 77 are shown in FIGS. 19 and 45; amino acid sequences offHbp ID 6 and fHbp ID 14 are provided above. In some embodiments, asubject fHbp comprises a single amino acid substitution (i.e., only oneamino acid substitution) relative to fHbp ID 1. In some embodiments, asubject fHbp comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions relative to a reference fHbp. In some embodiments, asubject fHbp comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acidsubstitutions relative to fHbp ID 1. In some embodiments, a subject fHbpcomprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutionsrelative to a naturally-occurring fHbp (e.g., fHbp ID 6, fHbp ID 14,fHbp ID 15, fHbp ID 28, as shown in FIG. 19, or anothernaturally-occurring fHbp). In some embodiments, a subject fHbp comprises2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions relative to theamino acid sequence of one of fHbp ID 1, fHbp ID 15, fHbp ID 22, fHbp ID28, and fHbp ID 77.

The amino acid residue position at which an alteration is introduced canbe determined by comparing the amino acid sequences of low fH binders(e.g. fHbp ID 14 and/or fHbp ID 15) with fHbps of a comparable affinityfor human fH as fHbp ID 1, for example. FHbps of a comparable affinityfor human fH as fHbp ID 1 or higher are referred herein as “high fHbinders”. Some examples of high fH binders include fHbp ID 1, fHbp ID 6,and fHbp ID 28. The low fH binders and high fH binders that share one ormore progenitor segments can be compared in a sequence alignment. SeeFIG. 19 as an example of a sequence alignment for determining amino acidalterations that can be made to a naturally-occurring fHbp in order toarrive at the subject fHbp.

A subject fHbp variant can be derived from (e.g., can include one ormore amino acid substitutions relative to) a variant 1 fHbp, a variant 2fHbp, or a variant 3 fHbp. A subject fHbp variant can be derived from(e.g., can include one or more amino acid substitutions relative to) amodular group I fHbp, a modular group II fHbp, a modular group III fHbp,a modular group IV fHbp, a modular group V fHbp, a modular group VIfHbp, a modular group VII fHbp, a modular group VIII fHbp, a modulargroup IX fHbp, or a modular group X fHbp.

Amino acid substitutions compared to a reference fHbp that are likely toresult in a fHbp with reduced affinity for fH include amino acidsubstitutions of fHbp amino acids that are contact residues for bindingto fH; amino acid substitutions of fHbp amino acids that are surfaceexposed; amino acid substitutions of fHbp amino acids at the interfacebetween the amino-terminal and carboxyl-terminal domains; and amino acidsubstitutions of fHbp amino acids that are proximal to a fH bindingresidue, where an amino acid that is “proximal to” an fH-binding aminoacid is an amino acid that is from one to ten residues amino-terminal toor carboxyl-terminal to the fH-binding amino acid. In certainembodiments, the amino acid substitution that results in a low affinityfor fH is not a fH contact residue. Certain contact residues are shownas bolded in FIG. 19.

Where a fHbp contains an amino acid substitution relative to anaturally-occurring or relative to fHbp ID1, the amino acid substitutioncan be conservative relative to that amino acid substitution. Forexample, if R41 is modified to S, making an R41S substitution, andparticularly where the R41S substitution results in a reduced affinityfor human fH, the present disclosure contemplates conservative aminoacid substitutions relative to S, such that the amino acid substitutionR41T is also contemplated.

The present disclosure provides a non-naturally occurring fHbp having anamino acid substitution at position 41 relative to fHbp ID 1, where theamino acid substitution is of a structure that disrupts interaction offHbp with human factor H, but provides that the mutant fHbp retainsimmunogenicity Amino acids suitable for substitution at position 41relative to fHbp ID1 include hydrophobic residues (e.g. Gly, Ala, Val,Leu, Ile, Pro); small polar residues (e.g. Ser, Cys, Thr, Met, Asn,Gln); small charged residues (e.g. Asp, Glu); and large hydrophobicresidues (e.g. Phe, Trp). Examples of substitutions that are predictednot to significantly disrupt interaction of fHbp with human factor H,and thus are to be avoided, include: large, charged residues (e.g. Lys).

In some embodiments, amino acid substitutions at one or more of thefollowing residues are specifically excluded: R41, Q38, Q87, Q113, K113,K119, D121, G121, Q126, Q128, R130, D201, E202, E218, A235, E239, andK241. In some embodiments, e.g., where a subject fHbp comprises a singleamino acid substitution relative to a reference fHbp (e.g., where thereference fHbp is a naturally-occurring fHbp or is fHbp ID 1), thesingle amino acid substitution can be at position E218 or E239.

Amino acid alterations found in the subject fHbps encompass those shownas shaded residues and/or boxed in FIG. 19 and listed below. In anexample of sequence analysis, segment A (V_(A); residues 8-73) of lowbinder fHbp ID 15 is compared to the V_(A) of the same progenitorsequence in a high fH binder, fHbp ID 28. As such, V_(A) segments fromboth fHbp ID 15 and fHbp ID 28 are identical in amino acid sequenceexcept at residue positions 41 and 60. As seen in FIG. 19, fHbp 15 hasS41 and R60 in V_(A); fHbp ID 28 has P41 and K60. Based on thisanalysis, amino acid residue positions corresponding to 41 and 60 offHbp ID 15 are candidate positions at which alteration can be introducedto arrive at the subject fHbp. In other words, a reference fHbp thatdoes not have S and R at residue positions corresponding to 41 and 60 offHbp ID 15, respectively, can be mutated to have S and/or R at positionscorresponding to 41 and 60 of fHbp ID 15. The subject fHbp comprisingone or more amino acid substitutions may then have lower affinity forhuman fH than without the substitutions (e.g. S41P, S41A, R41P, orR41A). Such fHbps are encompassed by the subject fHbps and are useful asimmunogen in eliciting bactericidal antibodies in subjects in needthereof.

Additional candidate residue positions at which an amino acid alterationcan be introduced are discussed in the examples below. For example, afHbp of the present disclosure can have an amino acid substitution atone or more positions corresponding to one or more amino acid residuesselected from 41, 60, 114, 113, 117, 119, 121, 128, 130, 147, 148, 149,178, 195, 218, 239, 241, or 247 of fHbp ID 1 (e.g., based on thenumbering of mature fHbp ID1. A fHbp of the present disclosure can havean amino acid substitution at one or more positions corresponding to oneor more amino acid residues selected from 41, 60, 80, 113, 114, 117,119, 121, 128, 130, 147, 148, 149, 178, 195, 199, 211, 220, 222, 236,241, 247, or 248, based on the numbering of the mature fHbp ID 1. A fHbpof the present disclosure can have an amino acid substitution at one ormore positions corresponding to one or more amino acid residues selectedfrom 87, 109, 115, 118, 126, 138, 197, 201, 202, 203, 209, 217, 225,235, or 245, based on the numbering of the mature fHbp ID 1. Where thecorresponding fHbp is a variant 2 or variant 3 fHbp (or a respectivecorresponding modular group), the modification can be introduced atposition 113, 119, and/or 121, or any combinations thereof. For example,a variant 2 fHbp (e.g. fHbp ID 77) may contain a substitution at one ormore positions at 113, 119, and/or 121, as well as a serine substitutionat position 41, or another suitable substitution at position 41 asdescribed above. Where the corresponding fHbp is an ID 22 variant, themodification can be introduced at position 80, 211, 218, 221, 223, 236,or 248, or any combination thereof.

A variant factor H binding protein (fHbp) of the present disclosure canalso have an amino acid substitution at one or more positionscorresponding to one or more amino acid residues selected from 60, 114,117, 147, 148, 149, 178, 195, or 247 of fHbp ID 1. Other positions canbe identified using sequence alignment studies between low fH bindersand high fH binders, similar to the one discussed above for V_(A) offHbp ID 15.

A variant fHbp of the present disclosure can be a variant of fHbp ID 1and can include one, two, three, or four of the following substitutions:R41S, R41A, R130A, H119A, E218A, and E239A. As discussed above, avariant fHbp of the present disclosure can include a single amino acidsubstitution. A variant fHbp of the present disclosure can also includea double amino acid substitution. For example, variant fHbp of thepresent disclosure can include substitutions at two of R41S, R41A,R130A, H119A, E218A, and E239A.

A variant fHbp of the present disclosure can have an amino acidsubstitution at one or more positions corresponding to one or more aminoacid residues selected from 80, 211, 218, 220, 222, 236, and 248 of fHbpID 1. Corresponding positions in fHbp variants are readilyascertainable, e.g., from the alignments presented in FIGS. 19, 34, and35. As non-limiting examples, a variant fHbp of the present disclosurecan be a variant of fHbp ID 22 and can include one, two, three, or fourof the following substitutions: R80A, D211A, E218A, T221A, H223A, G236I,and E248A. As discussed above, a variant fHbp of the present disclosurecan include a single amino acid substitution. A variant fHbp of thepresent disclosure can also include a double amino acid substitution.For example, variant fHbp of the present disclosure can includesubstitutions at two of R80, D211, E218, T221, H223, G236, and E248. Asone non-limiting example, a variant fHbp can include a T221A/H223Adouble substitution.

A variant fHbp of the present disclosure can have an amino acidsubstitution at one or more positions corresponding to one or more aminoacid residues selected from residues 41, 113, 119, 121 of fHbp ID 77. Asnon-limiting examples, a variant fHbp of the present disclosure can be avariant of fHbp ID 77 and can include one, two, three, or four of thefollowing substitutions: R41S, K113A, K119A, and D121A. As discussedabove, a variant fHbp of the present disclosure can include a singleamino acid substitution. A variant fHbp of the present disclosure canalso include a double amino acid substitution. For example, variant fHbpof the present disclosure can include substitutions at two of R41S,K113A, K119A, and D121A. As one non-limiting example, a variant fHbp caninclude a R41S/K113A double substitution, a R41S/K119A doublesubstitution, or a R41S/D121A double substitution.

A variant fHbp of the present disclosure can have an amino acidsubstitution at one or more positions corresponding to one or more aminoacid residues selected from residues 113, 121, 199, and 218 of fHbp IDID 28.

Where position R41 is substituted with serine in the fHbp of the presentdisclosure, its corresponding fHbp can belong to one of the modulargroups shown in FIG. 16. For example, the corresponding fHbp may be froma modular group I where all variable segments are of the α lineage.Examples of such subject fHbps include R41S mutants of fHbp IDs 1, 4, 9,and 94. In some embodiments, the subject fHbps do not include mutantsthat do not have a decreased affinity for human fH relative to theircorresponding fHbps. For example, the subject fHbps do not include R41Smutants of fHbp IDs 19 and 22.

Chimeric fHbps

As noted above, one or more modifications may be introduced into anaturally-occurring fHbp or a man-made fHbp (e.g. man-made chimericfHbp). The modification can encompass a modification in one segment orone domain while the other segments and/or domains may be derived fromany fHbp (e.g. a naturally-occurring fHbp of a different variant group).

In a fHbp described as having a modular architecture of V_(A), V_(B),V_(C) V_(D), and V_(E) segments, the modification can be introduced intoV_(A) of an α lineage (e.g. R41S in V_(A) of fHbp ID 1) while the othersegments of the fHbp (e.g. V_(B), V_(C) V_(D), and V_(E)) may each beindependently derived from any lineage, any variant groups, or any fHbpID. In another example, V_(A), V_(C), and V_(E) segments of a subjectfHbp can be derived from the α lineage (lineage 1) while V_(B), andV_(D) may be of a β lineage. Where the modification is a substitution ofarginine at position 41 with serine, the modification is introduced intoV_(A) of an α progenitor (V_(A)α). The V_(A) segment refers to acontiguous amino acid sequence that starts at residue position 7 andends at residue position 73, in which the position number corresponds tothose of the reference sequence, fHbp ID 1.

A fHbp of the present disclosure may contain an R41S mutation in a VAasegment containing an amino acid sequence that is at least about 90%, atleast about 92%, at least about 94%, at least about 95%, at least about96%, at least about 98%, at least about 99%, up to 100% identical to thefollowing sequence:

VAADIGAGLA DALTAPLDHK DKSLQSLTLD QSVRKNEKLK LAAQGAEKTY GNGDSLN TGKLKNDKV(SEQ ID NO:17). V_(A)α sequence is shown here with the R41S mutationbolded. A fHbp containing the modification of R41S thus has the R41Smutation in a V_(A)α segment and may have V_(B), V_(C) V_(D), and V_(E)segments, each independently derived from any other fHbp (e.g. adifferent lineage, a different variant group, or mutants of fHbp).

A chimeric fHbp of the present disclosure may also be described ashaving a modification in the N-terminal domain (fHbpN) of the fHbp whilethe C-terminal domain (fHbpC) may be derived from a different fHbp (e.g.a different variant group or a different lineage). “fHbpN” refers to acontiguous amino acid sequence that starts at about residue position 8and ends at about residue position 136. “fHbpC” refers to a contiguousamino acid sequence that starts at about residue position 141 and endsat about residue position 255. Intervening sequence between fHbpN andfHbpC is a linker between the two domains. As an example, the fHbpN of asubject fHbp can contain an R41S mutation in a sequence derived fromfHbp ID 1 while the fHbpC is derived from variant 2 or variant 3 fHbp(e.g. fHbp ID 77).

The corresponding chimeric fHbp may be of any known man-made chimeric,such as those described in Beernink et al. (2008) Infec. Immun.76:2568-2575 and WO 2009/114485, disclosure of which is incorporatedherein by reference. The chimeric containing the modification has adecreased affinity for human fH relative to the corresponding chimericfHbp, while still maintaining epitopes important for elicitingbactericidal response, such as those found in the corresponding chimericfHbp. fHbp epitopes that may be maintained in the modified chimericincludes those that are found in the corresponding chimeric fHbp such asthose described in WO 2009/114485, disclosure of which is incorporatedherein by reference. For example, a modified chimeric fHbp can containepitopes important for eliciting bactericidal antibody response againststrains containing variant 1 fHbp (e.g. epitopes in the N-terminaldomain such as those defined by mAb JAR 4 and/or JAR 5) and/or againststrains containing variant 2 or 3 fHbp (e.g. epitopes defined by mAb JAR10, JAR 11, JAR 13, and/or JAR 36). For example, the R41S mutation is amodification that can be introduced into the chimeric fHbp shown inFIGS. 19 and 45 in order to decrease binding to human fH while stillmaintaining JAR 4 and JAR 5 epitopes.

One feature of a subject fHbp is that when administered to a host (e.g.mammals such as mice or human), the subject fHbp can elicit abactericidal response at a level comparable or higher than thebactericidal response elicited by fHbp ID 1, or other correspondingreference (e.g. fHbp ID 4, 9, 22, 28, 74, or 77). Methods fordetermining levels of bactericidal response are known in the art anddescribed in the Example section below. For example, the geometric meanbactericidal titers of mice immunized with the subject fHbp is at leastabout 70%, at least about 80%, at least about 85%, at least about 90%,at least about 95%, at least about 100%, at least about 110%, at leastabout 120%, at least about 150%, at least about 175%, at least about200%, or more than 200%, of the geometric mean bactericidal titers ofmice immunized with fHbp ID 1. In some instances, the geometric meanbactericidal titer of a mouse immunized with a subject fHbp is at least2-fold, at least 2.5-fold, at least 5-fold, at least 10-fold, or morethan 10-fold, higher than the geometric mean bactericidal titer of acontrol mouse immunized with fHbp ID 1.

The subject fHbps can exclude those that elicit a bactericidal responsesignificantly lower than that elicited by fHbp ID 1. The subject fHbpscan exclude fHbp that have mutations at both residue positions 218 and239 (e.g. double mutant E218A/E239A). In some embodiments, the subjectfHbps encompass only non-naturally occurring fHbps; as such, in someembodiments, a subject fHbp excludes naturally occurring fHbps.

In many cases, a subject fHbp variant maintains and presents aconformational epitope bound by bacteridal antibodies that havebactericidal activity toward one or more Neisseria meningitidis strains.Thus, such fHbp mutants may maintain an epitope found in anaturally-occurring fHbp, while exhibiting reduced binding to fHcompared to the binding affinity for fH of a naturally-occurring fHbp.Variants that have minimal or no effect on the conformation of fHbp suchthat the mutant vaccine elicits bactericidal antibodies are consideredgood vaccine candidates. Whether a variant has an effect on theconformation of fHbp can be determined in various ways, includingbinding of antibodies listed in Table 9.

The fHbps of the present disclosure may have additional features,described in more detail below.

Conjugates

The subject fHbps of the present disclosure may contain one or moreadditional elements at the N- and/or C-terminus of the polypeptide, suchas a polypeptide (e.g. having an amino acid sequence heterologous to thesubject fHbp) and/or a carrier molecule. The additional heterologousamino acid sequences may be fused, e.g., to provide an N-terminalmethionine or derivative thereof (e.g., pyroglutamate) as a result ofexpression in a bacterial host cell (e.g., E. coli) and/or to provide achimeric polypeptide having a fusion partner at its N-terminus orC-terminus. Fusion partners of interest include, for example,glutathione-S-transferase (GST), maltose binding protein (MBP),His₆-tag, and the like, as well as leader peptides from other proteins,particularly lipoproteins. Fusion partners can provide for additionalfeatures, such as in facilitating isolation, purification, detection,immunogenicity of the subject fHbp.

Other elements that may be linked to the subject fHbp include a carriermolecule (e.g., a carrier protein, e.g. keyhole limpet hemocyanin(KLH)). Additional elements may be linked to the peptide via a linker,e.g. a flexible linker. Carriers encompass immunomodulators, a moleculethat directly or indirectly modifies an immune response. A specificclass of immunomodulators includes those that stimulate or aid in thestimulation of an immunological response. Examples include antigens andantigen carriers such as a toxin or derivative thereof, includingtetanus toxoid. Other carrier molecules that facilitate administrationand/or to increase the immunogenicity in a subject to be vaccinated ortreated against N. meningitidis are also contemplated. Carrier moleculescan also facilitate delivery to a cell or tissue of interest. Theadditional moiety may also aid in immunogenicity or forming a complexwith a component in a vaccine. The carrier molecules may act as ascaffold protein to facilitate display of the epitopes on a membranesurface (e.g. a vesicle vaccine).

In one example, the subject fHbps are modified at the N- and/orC-terminus to include a fatty acid (e.g. aliphatic carboxylic acidgroup). The fatty acid may be covalently linked to the fHbp via aflexible linker. An example of a fatty acid that may be used to modifyan end (e.g. N-terminal end, e.g., at the N-terminus) of the subjectfHbp is lauric acid. Lauric acid when covalently attached to anothermolecule is referred to as a lauroyl group (e.g. lauroyl sulfate).Lauric acid contains twelve carbon atoms with ten methylene groups andthe formula CH₃—(CH₂)₁₀—COOH. Other fatty acids that may be linked tothe subject peptides include caprylic acid (10 C), myristic acid (14 C),and palmitic acid (16 C). For details, see Westerink M A et al. (1995)Proc. Nall. Acad. Sci. USA 92:4021-4025. It is also contemplated thatany hydrophobic moiety that can serve to anchor the subject fHbp intothe bacterial outer membrane is contemplated herein for conjugation to aN- and/or C-terminal end (e.g., at the N-terminus) of the fHbps of thepresent disclosure, where the hydrophobic moiety can be optionallyconjugated to the peptide through a linker, e.g., a flexible linker, asdescribed herein. For example, a hydrophobic pentapeptide FLLAV (SEQ IDNO:18), as described in Lowell G H et al. (1988) J. Exp. Med.167:658-63.

As noted above, one way in which the fatty acid, as well as otheradditional elements described above, is connected to the fHbp is via alinker (e.g. lauroyl-Gly-Gly). Linkers suitable for use in modifying thefHbp of the present disclosure include “flexible linkers”. Suitablelinkers can be readily selected and can be of any of a suitable ofdifferent lengths, such as from 1 amino acid (e.g., Gly) to 20 aminoacids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.

Examples of flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), GSGGS_(n)(SEQ ID NO:19) and GGGS_(n) (SEQ ID NO:20), where n is an integer of atleast one), glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers known in the art. Glycine and glycine-serine polymersare of interest since both of these amino acids are relativelyunstructured, and therefore may serve as a neutral tether betweencomponents. Glycine polymers are of particular interest since glycineaccesses significantly more Ramachandran (or phi-psi) space than evenalanine, and are much less restricted than residues with longer sidechains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).Exemplary flexible linkers include, but are not limited GGSG (SEQ IDNO:21), GGSGG (SEQ ID NO:22), GSGSG (SEQ ID NO:23), GSGGG (SEQ IDNO:24), GGGSG (SEQ ID NO:25), GSSSG (SEQ ID NO:26), and the like. Theordinarily skilled artisan will recognize that design of a fHbpconjugated to any elements described above can include linkers that areall or partially flexible, such that the linker can include a flexiblelinker as well as one or more portions that confer less flexiblestructure.

Native fHbp usually contains an N-terminal cysteine to which a lipidmoiety can be covalently attached. This cysteine residue is usuallylipidated in the naturally-occurring protein, and can be lipidated inthe subject fHbps disclosed herein. Thus, in the amino acid sequencesdescribed herein, reference to “cysteine” or “C” at this positionspecifically includes reference to both an unmodified cysteine as wellas to a cysteine that is lipidated (e.g., due to post-translationalmodification). Thus, the subject fHbp can be lipidated or non-lipidated.Methods for production of lipidated proteins in vitro, (see, e.g.,Andersson et al. (2001) J. Immunological Methods 255:135-48) or in vivoare known in the art. For example, lipidated fHbp previously has beenpurified from the membrane fraction of E. coli protein by detergentextraction (Fletcher et al. (2004) Infection and Immunity 72:2088-100),which method may be adapted for the production of lipidated fHbp.Lipidated proteins may be of interest as such can be more immunogenicthan soluble protein (see, e.g., Fletcher et al. (2004) Infection andImmunity 72:2088-100).

It will be appreciated that the nucleotide sequences encodingheterologous fHbps can be modified so as to optimize the codon usage tofacilitate expression in a host cell of interest (e.g., E. coli, N.meningitidis, human (as in the case of a DNA-based vaccine), and thelike). Methods for production of codon optimized sequences are known inthe art.

Nucleic Acids Encoding fHbp

The present disclosure provides a nucleic acid encoding a subject fHbp.A subject nucleic acid will in some embodiments be present in arecombinant expression construct. Also provided are genetically modifiedhost cells comprising a subject nucleic acid.

fHbp polypeptides, and encoding nucleic acids of the present disclosurecan be derived from any suitable N. meningitidis strain. As is known inthe art, N. meningitidis strains are divided into serologic groups(capsular groups), serotypes (PorB phenotypes) and subtypes (PorAphenotypes) on the basis of reactions with polyclonal (Frasch, C. E. andChapman, 1973, J. Infect. Dis. 127: 149-154) or monoclonal antibodiesthat interact with different surface antigens. Capsular groupingtraditionally has been based on immunologically detectable variations inthe capsular polysaccharide but is being replaced by PCR of genesencoding specific enzymes responsible for the biosynthesis of thestructurally different capsular polysaccharides. About 12 capsulargroups (including A, B, C, X, Y, Z, 29-E, and W-135) are known. Strainsof the capsular groups A, B, C, Y and W-135 account for nearly allmeningococcal disease. Serotyping traditionally has been based onmonoclonal antibody defined antigenic differences in an outer membraneprotein called Porin B (PorB). Antibodies defining about 21 serotypesare currently known (Sacchi et al., 1998, Clin. Diag. Lab. Immunol.5:348). Serosubtyping has been based on antibody defined antigenicvariations on an outer membrane protein called Porin A (PorA). Bothserotyping and serosubtyping are being replaced by PCR and/or DNAsequencing for identification of genes encoding the variable regions ofPorB and PorA, respectively that are associated with mAb reactivity(e.g. Sacchi, Lemos et al., supra; Urwin et al., 1998, Epidem. andInfect. 120:257).

While N. meningitidis strains of any capsular group may be used, N.meningitidis strains of capsular group B can be sources from whichnucleic acid encoding fHbp and domains thereof are derived.

Nucleic acids encoding fHbp polypeptides for use in construction of thesubject fHbps contemplated herein are known in the art. Various fHbp andtheir sequences are available at neisseria.org andpubmlst.org/neisseria/fHbp websites. Examples of fHbp polypeptides arealso described in, for example, U.S. patent application No. 61/174,424,PCT application number PCT/US09/36577, WO 2004/048404; Masignani et al.(2003) J Exp Med 197:789-799; Fletcher et al. (2004) Infect Immun72:2088-2100; Welsch et al. J Immunol 2004 172:5606-5615; and WO99/57280. Nucleic acid (and amino acid sequences) for fHbp variants andsubvariants are also provided in GenBank as accession nos.: NC_003112,GeneID: 904318 (NCBI Ref. NP_274866), fHbp ID 1 from N. meningitidisstrain MC58; AY548371 (AAT01290.1) (from N. meningitidis strain CU385);AY548370 (AAT01289.1) (from N. meningitidis strain H44/76); AY548377(AAS56920.1) fHbp ID 4 from N. meningitidis strain M4105; AY548376(AAS56919.1) (from N. meningitidis strain M1390); AY548375 (AAS56918.1)(from N. meningitidis strain NZ98/254); AY548374 (AAS56917.1) (from N.meningitidis strain M6190); AY548373 (AAS56916.1) (from N. meningitidisstrain 4243); and AY548372 (AAS56915.1) (from N. meningitidis strainBZ83).

For purposes of identifying relevant amino acid sequences contemplatedfor use in the subject fHbps disclosed herein, it should be noted thatthe immature fHbp includes a leader sequence of about 19 residues.Furthermore, when provided an amino acid sequence the ordinarily skilledperson can readily envision the sequences of nucleic that can encodefor, and provide for expression of, a polypeptide having such an aminoacid sequence.

In addition to the specific amino acid sequences and nucleic acidsequences provided herein, the disclosure also contemplates polypeptidesand nucleic acids having sequences that are at least 80%, at least 85%,at least 90%, or at least 95% identical in sequence to such examples ofamino acid and nucleic acids. The terms “identical” or percent“identity,” in the context of two or more polynucleotide sequences, ortwo or more amino acid sequences, refers to two or more sequences orsubsequences that are the same or have a specified percentage of aminoacid residues or nucleotides that are the same (e.g., at least 80%, atleast 85%, at least 90%, or at least 95% identical over a specifiedregion), when compared and aligned for maximum correspondence over adesignated region, e.g., a Y_(E) or a region of at least about 40, 45,50, 55, 60, 65 or more amino acids or nucleotides in length, and can beup to the full-length of the reference amino acid or nucleotide sequence(e.g., a full-length fHbp). The disclosure specifically contemplatesboth naturally-occurring polymorphisms and synthetically produced aminoacid sequences and their encoding nucleic acids.

For sequence comparison, typically one sequence acts as a referencesequence (e.g., a naturally-occurring fHbp polypeptide sequence or asegment thereof), to which test sequences are compared. When using asequence comparison algorithm, test and reference sequences are inputinto a computer program, sequence coordinates are designated, ifnecessary, and sequence algorithm program parameters are designated. Thesequence comparison algorithm then calculates the percent sequenceidentity for the test sequence(s) relative to the reference sequence,based on the designated program parameters.

Examples of algorithms that are suitable for determining percentsequence identity are the BLAST and BLAST 2.0 algorithms, which aredescribed in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 andAltschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information (www.ncbi.nlm.nih.gov).Further exemplary algorithms include ClustalW (Higgins D., et al. (1994)Nucleic Acids Res 22: 4673-4680), available atwww.ebi.ac.uk/Tools/clustalw/index.html.

Some residue positions which are not identical differ by conservativeamino acid substitutions. Conservative amino acid substitutions refer tothe interchangeability of residues having similar side chains. Forexample, a group of amino acids having aliphatic side chains is glycine,alanine, valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having acidic sidechains is aspartate and glutamate; a group of amino acids having basicside chains is lysine, arginine, and histidine; and a group of aminoacids having sulfur-containing side chains is cysteine and methionine.

Sequence identity between two nucleic acids can also be described interms of hybridization of two molecules to each other under stringentconditions. The hybridization conditions are selected following standardmethods in the art (see, for example, Sambrook, et al., MolecularCloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor,N.Y.). An example of stringent hybridization conditions is hybridizationat 50° C. or higher and 0.1×SSC (15 mM sodium chloride/1.5 mM sodiumcitrate). Another example of stringent hybridization conditions isovernight incubation at 42° C. in a solution: 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65° C. Stringent hybridization conditions are hybridizationconditions that are at least as stringent as the above representativeconditions, where conditions are considered to be at least as stringentif they are at least about 80% as stringent, typically at least 90% asstringent as the above specific stringent conditions.

Methods of Production

The fHbps of the present disclosure can be produced by any suitablemethod, including recombinant and non-recombinant methods (e.g.,chemical synthesis). Where the subject fHbp is produced usingrecombinant techniques, the methods can involve any suitable constructand any suitable host cell, which can be a prokaryotic or eukaryoticcell, usually a bacterial or yeast host cell, more usually a bacterialcell. Methods for introduction of genetic material into host cellsinclude, for example, transformation, electroporation, conjugation,calcium phosphate methods and the like. The method for transfer can beselected so as to provide for stable expression of the introducedfHbp-encoding nucleic acid. The fHbp-encoding nucleic acid can beprovided as an inheritable episomal element (e.g., plasmid) or can begenomically integrated.

Suitable vectors for transferring fHbp-encoding nucleic acid can vary incomposition. Integrative vectors can be conditionally replicative orsuicide plasmids, bacteriophages, and the like. The constructs caninclude various elements, including for example, promoters, selectablegenetic markers (e.g., genes conferring resistance to antibiotics (forinstance kanamycin, erythromycin, chloramphenicol, or gentamycin)),origin of replication (to promote replication in a host cell, e.g., abacterial host cell), and the like. The choice of vector will dependupon a variety of factors such as the type of cell in which propagationis desired and the purpose of propagation. Certain vectors are usefulfor amplifying and making large amounts of the desired DNA sequence.Other vectors are suitable for expression in cells in culture. Stillother vectors are suitable for transfer and expression in cells in awhole animal. The choice of appropriate vector is well within the skillof the art. Many such vectors are available commercially.

In one example, the vector is an expression vector based on episomalplasmids containing selectable drug resistance markers and elements thatprovide for autonomous replication in different host cells (e.g., inboth E. coli and N. meningitidis). One example of such a “shuttlevector” is the plasmid pFP10 (Pagotto et al. (2000) Gene 244:13-19).

Constructs can be prepared by, for example, inserting a polynucleotideof interest into a construct backbone, typically by means of DNA ligaseattachment to a cleaved restriction enzyme site in the vector.Alternatively, the desired nucleotide sequence can be inserted byhomologous recombination or site-specific recombination. Typicallyhomologous recombination is accomplished by attaching regions ofhomology to the vector on the flanks of the desired nucleotide sequence,while site-specific recombination can be accomplished through use ofsequences that facilitate site-specific recombination (e.g., cre-lox,att sites, etc.). Nucleic acid containing such sequences can be addedby, for example, ligation of oligonucleotides, or by polymerase chainreaction using primers comprising both the region of homology and aportion of the desired nucleotide sequence.

Vectors can provide for extrachromosomal maintenance in a host cell orcan provide for integration into the host cell genome. Vectors are amplydescribed in numerous publications well known to those in the art,including, e.g., Short Protocols in Molecular Biology, (1999) F.Ausubel, et al., eds., Wiley & Sons. Vectors may provide for expressionof the nucleic acids encoding the subject fHbp, may provide forpropagating the subject nucleic acids, or both.

Examples of vectors that may be used include but are not limited tothose derived from recombinant bacteriophage DNA, plasmid DNA or cosmidDNA. For example, plasmid vectors such as pBR322, pUC 19/18, pUC 118,119 and the M13 mp series of vectors may be used. pET21 is also anexpression vector that may be used. Bacteriophage vectors may includeλgt10, λgt11, λgt18-23, λZAP/R and the EMBL series of bacteriophagevectors. Further vectors that may be utilized include, but are notlimited to, pJB8, pCV 103, pCV 107, pCV 108, pTM, pMCS, pNNL, pHSG274,COS202, COS203, pWE15, pWE16 and the charomid 9 series of vectors.

For expression of a subject fHbp, an expression cassette may beemployed. Thus, the present disclosure provides a recombinant expressionvector comprising a subject nucleic acid. The expression vector providestranscriptional and translational regulatory sequences, and may providefor inducible or constitutive expression, where the coding region isoperably linked under the transcriptional control of the transcriptionalinitiation region, and a transcriptional and translational terminationregion. These control regions may be native to an fHbp from which thesubject fHbp is derived, or may be derived from exogenous sources. Ingeneral, the transcriptional and translational regulatory sequences mayinclude, but are not limited to, promoter sequences, ribosomal bindingsites, transcriptional start and stop sequences, translational start andstop sequences, and enhancer or activator sequences. Promoters can beeither constitutive or inducible, and can be a strong constitutivepromoter (e.g., T7, and the like).

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding proteins of interest. A selectable marker operativein the expression host may be present to facilitate selection of cellscontaining the vector. In addition, the expression construct may includeadditional elements. For example, the expression vector may have one ortwo replication systems, thus allowing it to be maintained in organisms,for example in mammalian or insect cells for expression and in aprokaryotic host for cloning and amplification. In addition theexpression construct may contain a selectable marker gene to allow theselection of transformed host cells. Selection genes are well known inthe art and will vary with the host cell used.

It should be noted that fHbps of the present disclosure may compriseadditional elements, such as a detectable label, e.g., a radioactivelabel, a fluorescent label, a biotin label, an immunologicallydetectable label (e.g., an HA tag, a poly-Histidine tag) and the like.Additional elements of fHbp can be provided to facilitate isolation(e.g., biotin tag, immunologically detectable tag) through variousmethods (e.g., affinity capture, etc.). The subject fHbp can optionallybe immobilized on a support through covalent or non-covalent attachment.

Isolation and purification of fHbp can be accomplished according tomethods known in the art. For example, fHbp can be isolated from alysate of cells genetically modified to express a fHbp, or from asynthetic reaction mix, by immunoaffinity purification, which generallyinvolves contacting the sample with an anti-fHbp antibody (e.g., ananti-fHbp mAb, such as a JAR 5 MAb or other appropriate JAR MAb known inthe art), washing to remove non-specifically bound material, and elutingspecifically bound fHbp. Isolated fHbp can be further purified bydialysis and other methods normally employed in protein purificationmethods. In one example, the fHbp can be isolated using metal chelatechromatography methods.

Host Cells

Any of a number of suitable host cells can be used in the production offHbp. In general, the fHbp described herein may be expressed inprokaryotes or eukaryotes, usually bacteria, more usually E. coli orNeisseria (e.g., N. meningitidis) in accordance with conventionaltechniques. Thus, the present disclosure further provides a geneticallymodified host cell, which contains a nucleic acid encoding a subjectfHbp. Host cells for production (including large scale production) of asubject fHbp can be selected from any of a variety of available hostcells. Examples of host cells for expression include those of aprokaryotic or eukaryotic unicellular organism, such as bacteria (e.g.,Escherichia coli strains), yeast (e.g., S. cerevisiae, Pichia spp., andthe like), and may include host cells originally derived from a higherorganism such as insects, vertebrates, particularly mammals, (e.g. CHO,HEK, and the like). Generally bacterial host cells and yeast are ofparticular interest for subject fHbp production.

Subject fHbps can be prepared in substantially pure or substantiallyisolated form (i.e., substantially free from other Neisserial or hostcell polypeptides) or substantially isolated form. The subject fHbp canbe present in a composition that is enriched for the polypeptiderelative to other components that may be present (e.g., otherpolypeptides or other host cell components). Purified subject fHbp canbe provided such that the polypeptide is present in a composition thatis substantially free of other expressed polypeptides, e.g., less than90%, usually less than 60% and more usually less than 50% of thecomposition is made up of other expressed polypeptides.

Host Cells for Vesicle Production

Where a subject fHbp is to be provided in a membrane vesicle (asdiscussed in more detail below), a Neisserial host cell is geneticallymodified to express a subject fHbp. Any of a variety of Neisseria spp.strains can be modified to produce a subject fHbp, and, optionally,which produce or can be modified to produce other antigens of interest,such as PorA, can be used in the methods disclosed herein.

Methods and vectors to provide for genetic modification of Neisserialstrains and expression of a desired polypeptide are known in the art.Examples of vectors and methods can be found in WO 02/09746 and O'Dwyeret al. (2004) Infect Immun 72:6511-80. Strong promoters, particularlyconstitutive strong promoters are of particular interest. Examples ofpromoters include the promoters of porA, porB, lbpB, tbpB, p110, hpuAB,lgtF, opa, p110, lst, hpuAB, and rmp.

Pathogenic Neisseria spp. or strains derived from pathogenic Neisseriaspp., particularly strains pathogenic for humans or derived from strainspathogenic or commensal for humans, are of particular interest for usein membrane vesicle production. Examples of Neisserial spp. include N.meningitidis, N. flavescens, N. gonorrhoeae, N. lactamica, N.polysaccharea, N. cinerea, N. mucosa, N. subflava, N. sicca, N.elongata, and the like.

N. meningitidis strains are of particular interest for geneticmodification to express the subject fHbps and for use in vesicleproduction. The strain used for vesicle production can be selectedaccording to a number of different characteristics that may be desired.For example, the strain may be selected according to: a desired PorAtype (a “serosubtype”, as described above), capsular group, serotype,and the like; decreased capsular polysaccharide production; and thelike. For example, the production strain can produce any desired PorApolypeptide, and may express one or more PorA polypeptides (eithernaturally or due to genetic engineering). Examples of strains includethose that produce a PorA polypeptide which confers a serosubtype ofP1.7,16; P1.19,15; P1.7,1; P1.5,2; P1.22a,14; P1.14; P1.5,10; P1.7,4;P1.12,13; as well as variants of such PorA polypeptides which may or maynot retain reactivity with conventional serologic reagents used inserosubtyping. Also of interest are PorA polypeptides characterizedaccording to PorA variable region (VR) typing (see, e.g., Russell et al.(2004) Emerging Infect Dis 10:674-678; Sacchi C T et al. (1998) ClinDiagn Lab Immunol 5:845-55; Sacchi et al (2000) J. Infect Dis182:1169-1176). A substantial number of distinct VR types have beenidentified, which can be classified into VR1 and VR2 family“prototypes”. A web-accessible database describing this nomenclature andits relationship to previous typing schemes is found atneisseria.org/nm/typing/pora. Alignments of certain PorA VR1 and VR2types are provided in Russell et al. (2004) Emerging Infect Dis10:674-678.

Alternatively or in addition, the production strain can be a capsuledeficient strain. Capsule deficient strains can provide vesicle-basedvaccines that provide for a reduced risk of eliciting a significantautoantibody response in a subject to whom the vaccine is administered(e.g., due to production of antibodies that cross-react with sialic acidon host cell surfaces). “Capsule deficient” or “deficient in capsularpolysaccharide” as used herein refers to a level of capsularpolysaccharide on the bacterial surface that is lower than that of anaturally-occurring strain or, where the strain is genetically modified,is lower than that of a parental strain from which the capsule deficientstrain is derived. A capsule deficient strain includes strains that aredecreased in surface capsular polysaccharide production by at least 10%,20%, 25%, 30%, 40%, 50%, 60%, 75%, 80%, 85%, 90% or more, and includesstrains in which capsular polysaccharide is not detectable on thebacterial surface (e.g., by whole cell enzyme-linked immunosorbent assay(ELISA) using an anti-capsular polysaccharide antibody).

Capsule deficient strains include those that are capsule deficient dueto a naturally-occurring or recombinantly-generated geneticmodification. Naturally-occurring capsule deficient strains (see, e.g.,Dolan-Livengood et al. (2003) J. Infect. Dis. 187:1616-28), as well asmethods of identifying and/or generating capsule-deficient strains (see,e.g., Fisseha et al. (2005) Infect. Immun. 73:4070-4080; Stephens et al.(1991) Infect Immun 59:4097-102; Frosch et al. (1990) Mol Microbiol.4:1215-1218) are known in the art.

Modification of a Neisserial host cell to provide for decreasedproduction of capsular polysaccharide may include modification of one ormore genes involved in capsule synthesis, where the modificationprovides for, for example, decreased levels of capsular polysacchariderelative to a parent cell prior to modification. Such geneticmodifications can include changes in nucleotide and/or amino acidsequences in one or more capsule biosynthesis genes rendering the straincapsule deficient (e.g., due to one or more insertions, deletions,substitutions, and the like in one or more capsule biosynthesis genes).Capsule deficient strains can lack or be non-functional for one or morecapsule genes.

Of particular interest are strains that are deficient in sialic acidbiosynthesis. Such strains can provide for production of vesicles thathave reduced risk of eliciting anti-sialic acid antibodies thatcross-react with human sialic acid antigens, and can further provide forimproved manufacturing safety. Strains having a defect in sialic acidbiosynthesis (due to either a naturally occurring modification or anengineered modification) can be defective in any of a number ofdifferent genes in the sialic acid biosynthetic pathway. Of particularinterest are strains that are defective in a gene product encoded by theN-acetylglucosamine -6-phosphate 2-epimerase gene (known as synXAAF40537.1 or siaA AAA20475), with strains having this gene inactivatedbeing of especial interest. For example, in one embodiment, a capsuledeficient strain is generated by disrupting production of a functionalsynX gene product (see, e.g., Swartley et al. (1994) J Bacteriol.176:1530-4).

Capsule-deficient strains can also be generated from naturally-occurringstrains using non-recombinant techniques, e.g., by use of bactericidalanti-capsular antibodies to select for strains with reduced levels ofcapsular polysaccharide.

Where the disclosure involves use of two or more strains (e.g., toproduce antigenic compositions containing a subject fHbp-presentingvesicles from different strains), the strains can be selected so as todiffer in one or more strain characteristics, e.g., to provide forvesicles that differ in the subject fHbp used, PorA, and the like.

Preparation of Vesicles

The antigenic compositions contemplated by the present disclosuregenerally include vesicles prepared from Neisserial cells that express asubject fHbp. As referred to herein “vesicles” is meant to encompassouter membrane vesicles as well as microvesicles (which are alsoreferred to as blebs).

The antigenic composition can contain outer membrane vesicles (OMV)prepared from the outer membrane of a cultured strain of Neisseriameningitidis spp. genetically modified to express a subject fHbp. OMVsmay be obtained from Neisseria meningitidis grown in broth or solidmedium culture, preferably by separating the bacterial cells from theculture medium (e.g. by filtration or by a low-speed centrifugation thatpellets the cells, or the like), lysing the cells (e.g. by addition ofdetergent, osmotic shock, sonication, cavitation, homogenization, or thelike) and separating an outer membrane fraction from cytoplasmicmolecules (e.g. by filtration; or by differential precipitation oraggregation of outer membranes and/or outer membrane vesicles, or byaffinity separation methods using ligands that specifically recognizeouter membrane molecules; or by a high-speed centrifugation that pelletsouter membranes and/or outer membrane vesicles, or the like); outermembrane fractions may be used to produce OMVs.

The antigenic composition can contain microvesicles (MV) (or “blebs”)containing subject fHbps, where the MV or blebs are released duringculture of a Neisseria meningitidis strain genetically modified toexpress a subject fHbp. For example, MVs may be obtained by culturing astrain of Neisseria meningitidis in broth culture medium, separatingwhole cells from the broth culture medium (e.g. by filtration, or by alow-speed centrifugation that pellets only the cells and not the smallerblebs, or the like), and then collecting the MVs that are present in thecell-free culture medium (e.g. by filtration, differential precipitationor aggregation of MVs, or by a high-speed centrifugation that pelletsthe blebs, or the like). Strains for use in production of MVs cangenerally be selected on the basis of the amount of blebs produced inculture (e.g., bacteria can be cultured in a reasonable number toprovide for production of blebs suitable for isolation andadministration in the methods described herein). An exemplary strainthat produces high levels of blebs is described in PCT Publication No.WO 01/34642. In addition to bleb production, strains for use in MVproduction may also be selected on the basis of NspA production, wherestrains that produce higher levels of NspA may be of particular interest(for examples of N. meningitidis strains having different NspAproduction levels, see, e.g., Moe et al. (1999 Infect. Immun 67: 5664).Other strains of interest for use in production of blebs include strainshaving an inactivated GNA33 gene, which encodes a lipoprotein requiredfor cell separation, membrane architecture and virulence (see, e.g.,Adu-Bobie et al. (2004) Infect Immun. 72:1914-1919).

The antigenic compositions of the present disclosure can containvesicles from one strain, or from 2, 3, 4, 5 or more strains, whichstrains may be homologous or heterologous, usually heterologous, to oneanother. For example, the strains may be homologous or heterologous withrespect to PorA and/or the fHbp from which the subject fHbp is derived.The vesicles can be prepared from strains that express more than onesubject fHbp (e.g., 1, 2, 3, or more subject fHbp) which may be composedof fHbp amino acid sequences from different variants (v.1, v.2, or v.3)or subvariants (e.g., a subvariant of v.1, v.2, or v.3).

The antigenic compositions can comprise a mixture of OMVs and MVspresenting the same or different subject fHbps, where the subject fHbpsmay optionally present epitopes from different combinations of fHbpvariants and/or subvariants and where the OMVs and/or MVs may be fromthe same or different strains. Vesicles from different strains can beadministered as a mixture, or can be administered serially.

Where desired (e.g., where the strains used to produce vesicles areassociated with endotoxin or particular high levels of endotoxin), thevesicles are optionally treated to reduce endotoxin, e.g., to reducetoxicity following administration. Although less desirable as discussedbelow, reduction of endotoxin can be accomplished by extraction with asuitable detergent (for example, BRIJ-96, sodium deoxycholate, sodiumlauroylsarcosinate, Empigen BB, TRITON X-100, TWEEN 20 (sorbitanmonolaurate polyoxyethylene), TWEEN 80, at a concentration of 0.1-10%,preferably 0.5-2%, and SDS). Where detergent extraction is used, it ispreferable to use a detergent other than deoxycholate.

The vesicles of the antigenic compositions can be prepared withoutdetergent, e.g., without use of deoxycholate. Although detergenttreatment is useful to remove endotoxin activity, it may deplete thenative fHbp lipoprotein and/or subject fHbp (including lipidated fHbp)by extraction during vesicle production. Thus it may be particularlydesirable to decrease endotoxin activity using technology that does notrequire a detergent. In one approach, strains that are relatively lowproducers of endotoxin (lipopolysaccharide, LPS) are used so as to avoidthe need to remove endotoxin from the final preparation prior to use inhumans. For example, the vesicles can be prepared from Neisseria mutantsin which lipooligosaccharide or other antigens that may be undesirablein a vaccine (e.g. Rmp) is reduced or eliminated.

Vesicles can be prepared from N. meningitidis strains that containgenetic modifications that result in decreased or no detectable toxicactivity of lipid A. For example, such strain can be geneticallymodified in lipid A biosynthesis (Steeghs et al. (1999) Infect Immun67:4988-93; van der Ley et al. (2001) Infect Immun 69:5981-90; Steeghset al. (2004) J Endotoxin Res 10:113-9; Fissha et al, (2005) InfectImmun 73:4070). The immunogenic compositions may be detoxified bymodification of LPS, such as downregulation and/or inactivation of theenzymes encoded by lpxL1 or lpxL2, respectively. Production of apenta-acylated lipid A made in lpxL1 mutants indicates that the enzymeencoded by lpxL1 adds the C12 to the N-linked 3-OH C14 at the 2′position of GlcN II. The major lipid A species found in lpxL2 mutants istetra-acylated, indicating the enzyme encoded by lpxL2 adds the otherC12, i.e., to the N-linked 3-OH C14 at the 2 position of GlcN I.Mutations resulting in a decreased (or no) expression of these genes (ordecreased or no activity of the products of these genes) result inaltered toxic activity of lipid A (van der Ley et al. (2001) InfectImmun 69:5981-90). Tetra-acylated (lpxL2 mutant) and penta acylated(lpxL1 mutant) lipid A are less toxic than the wild-type lipid A.Mutations in the lipid A 4′-kinase encoding gene (lpxK) also decreasethe toxic activity of lipid A. Of particular interest for use inproduction of vesicles (e.g., MV or OMV) are N. meningitidis strainsgenetically modified so as to provide for decreased or no detectablefunctional LpxL1-encoded protein, e.g., where the Neisseria bacterium(e.g., N. meningitidis strain) is genetically modified to provide fordecreased or no activity of a gene product of the lpxL1 gene. Forexample, the Neisseria bacterium can be genetically modified to have anlpxL1 gene knockout, e.g., where the lpxL1 gene is disrupted. See, e.g.,US Patent Publication No. 2009/0035328. The Neisseria bacterium can begenetically modified to provide for decreased or no activity of a geneproduct of the lpxL2 gene. The Neisseria bacterium can be geneticallymodified to provide for decreased or no activity of a gene product ofthe lpxL1 gene and the lpxL2 gene. Such vesicles provide for reducedtoxicity as compared to N. meningitidis strains that are wild-type forLPS production, while retaining immunogenicity of subject fHbp.

LPS toxic activity can also be altered by introducing mutations ingenes/loci involved in polymyxin B resistance (such resistance has beencorrelated with addition of aminoarabinose on the 4′ phosphate of lipidA). These genes/loci could be pmrE that encodes a UDP-glucosedehydrogenase, or a region of antimicrobial peptide-resistance genescommon to many enterobacteriaciae which could be involved inaminoarabinose synthesis and transfer. The gene pmrF that is present inthis region encodes a dolicol-phosphate manosyl transferase (Gunn J. S.,Kheng, B. L., Krueger J., Kim K., Guo L., Hackett M., Miller S. I. 1998.Mol. Microbiol. 27: 1171-1182).

Mutations in the PhoP-PhoQ regulatory system, which is a phospho-relaytwo component regulatory system (e.g., PhoP constitutive phenotype,PhoPc), or low Mg++ environmental or culture conditions (that activatethe PhoP-PhoQ regulatory system) lead to the addition of aminoarabinoseon the 4′-phosphate and 2-hydroxymyristate replacing myristate(hydroxylation of myristate). This modified lipid A displays reducedability to stimulate E-selectin expression by human endothelial cellsand TNF secretion from human monocytes.

Polymyxin B resistant strains are also suitable for use, as such strainshave been shown to have reduced LPS toxicity (see, e.g., van der Ley etal. (1994) In: Proceedings of the ninth international pathogenicNeisseria conference. The Guildhall, Winchester, England).Alternatively, synthetic peptides that mimic the binding activity ofpolymyxin B may be added to the antigenic compositions to reduce LPStoxic activity (see, e.g., Rustici et al. (1993) Science 259:361-365;Porro et al. (1998) Prog Clin Biol Res. 397:315-25).

Endotoxin can also be reduced through selection of culture conditions.For example, culturing the strain in a growth medium containing 0.1mg-100 mg of aminoarabinose per liter medium provides for reduced lipidtoxicity (see, e.g., WO 02/097646).

Compositions and Formulations

“Compositions”, “antigen composition”, “antigenic composition” or“immunogenic composition” is used herein as a matter of convenience torefer generically to compositions comprising a subject fHbp as disclosedherein, which subject fHbp may be optionally conjugated to furtherenhance immunogenicity. Compositions useful for eliciting antibodies,e.g., antibodies against Neisseria meningitidis, e.g., bactericidalantibodies to Neisseria meningitidis, in a human are specificallycontemplated by the present disclosure. Antigenic compositions cancontain 1, 2, or more different subject fHbps. Where there is more thanone type of fHbp, each subject fHbps may present epitopes from differentcombinations of fHbp variants and/or subvariants.

Antigenic compositions contain an immunologically effective amount of asubject fHbp, and may further include other compatible components, asneeded. Compositions of the present disclosure can contain fHbp that arelow fH binders. Low fH binders in the subject compositions encompass anyfHbp described above, such as non-naturally-occurring ornaturally-occurring fHbp (e.g. fHbp ID 14 and/or fHbp ID 15). Thecomposition contain one or more fHbp, in which at least one fHbp is alow fH binder. Where there is more than one fHbp in a composition, eachfHbp may be different (e.g. in amino acid sequences and/or conjugation).

Immunogenic compositions contemplated by the present disclosure include,but are not limited to, compositions comprising:

1) a non-naturally occurring fHbp (e.g., a non-naturally occurring fHbpthat has lower affinity for human fH than fHbp ID 1); and

2) a fHbp (e.g., a non-naturally occurring fHbp, e.g., a non-naturallyoccurring fHbp that has a lower affinity for fH than fHbp ID 1) andNspA;

where the fHbp and/or NspA can be provided as recombinant proteinsand/or in a vesicle-based composition (e.g., OMV). It should be notedthat where the composition includes both NspA and a fHbp, thebactericidal activity of antibodies elicited by administration of thecomposition can result from cooperation of antibodies to one or bothantigens. Examples of immunogenic compositions provided by the presentdisclosure include:

a) an immunogenic composition that comprises a non-naturally occurringfHbp variant as described above (e.g., a, where the non-naturallyoccurring fHbp elicits a bactericidal antibody response to at least oneNeisseria meningitidis strain);

b) an immunogenic composition that comprises a non-naturally occurringfHbp variant as described above (e.g., a non-naturally occurring fHbpthat has lower affinity for human fH than fHbp ID 1); and a recombinantNspA protein;

c) an immunogenic composition that comprises an isolated fHbp comprisingat least 85% amino acid sequence identity to fHbp ID 14 or fHbp ID 15,where the fHbp has lower affinity for human factor H (fH) than fHbp ID1;

d) an immunogenic composition that comprises an isolated fHbp comprisingat least 85% amino acid sequence identity to fHbp ID 14 or fHbp ID 15,where the fHbp has lower affinity for human factor H (fH) than fHbp ID1; and a recombinant NspA protein;

e) an immunogenic composition that comprises a native OMV obtained froma genetically modified Neisseria host cell that is genetically modifiedwith a nucleic acid encoding a non-naturally occurring fHbp variant asdescribed above (e.g., a non-naturally occurring fHbp that has loweraffinity for human fH than fHbp ID 1), such that the encodednon-naturally occurring fHbp is produced by the genetically modifiedhost cell, where the OMV comprises the encoded non-naturally occurringfHbp; and

f) an immunogenic composition that comprises a native OMV obtained froma genetically modified Neisseria host cell that is genetically modifiedwith a nucleic acid encoding a non-naturally occurring fHbp variant asdescribed above (e.g., a non-naturally occurring fHbp that has loweraffinity for human fH than fHbp ID 1, such that the encodednon-naturally occurring fHbp is produced by the genetically modifiedhost cell, where the OMV comprises the encoded non-naturally occurringfHbp; and where the Neisseria host cell also produces higher levels ofNspA, such that the OMV also comprises NspA. For example, the Neisseriahost cell can be one that is genetically modified for increasedexpression of NspA.

By “immunologically effective amount” is meant that the administrationof that amount to an individual, either in a single dose, as part of aseries of the same or different antigenic compositions, is effective toelicit an antibody response effective for treatment or prevention of asymptom of, or disease caused by, for example, infection by Neisseria,particularly N. meningitidis, more particularly Group B N. meningitidis.This amount varies depending upon the health and physical condition ofthe individual to be treated, age, the capacity of the individual'simmune system to produce antibodies, the degree of protection desired,the formulation of the vaccine, the treating clinician's assessment ofthe medical situation, and other relevant factors. It is expected thatthe amount will fall in a relatively broad range that can be determinedthrough routine trials.

Amino acid sequences of NspA polypeptides are known in the art. See,e.g., WO 96/29412; and Martin et al. (1997) J. Exp. Med. 185:1173;GenBank Accession No. U52066; and GenBank Accession No. AAD53286. An“NspA polypeptide” can comprise an amino acid sequence having at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,at least about 98%, at least about 99%, or 100%, amino acid sequenceidentity to a contiguous stretch of from about 75 amino acids to about100 amino acids, from about 100 amino acids to about 150 amino acids orfrom about 150 amino acids to about 174 amino acids, of the amino acidsequence depicted in FIG. 44. An “NspA polypeptide” can comprise anamino acid sequence having at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98%, at least about99%, or 100%, amino acid sequence identity to a contiguous stretch offrom about 75 amino acids to about 100 amino acids, or from about 100amino acids to about 155 amino acids, of amino acids 20 to 174 of theamino acid sequence depicted in FIG. 44. In some cases, the NspApolypeptide lacks a signal sequence; in other cases (e.g., forexpression in a host cell), the NspA polypeptide includes a signalsequence.

Dosage regimen may be a single dose schedule or a multiple dose schedule(e.g., including booster doses) with a unit dosage form of the antigeniccomposition administered at different times. The term “unit dosageform,” as used herein, refers to physically discrete units suitable asunitary dosages for human and animal subjects, each unit containing apredetermined quantity of the antigenic compositions of the presentdisclosure in an amount sufficient to produce the desired effect, whichcompositions are provided in association with a pharmaceuticallyacceptable excipient (e.g., pharmaceutically acceptable diluent, carrieror vehicle). The antigenic composition may be administered inconjunction with other immunoregulatory agents.

Antigenic compositions can be provided in a pharmaceutically acceptableexcipient, which can be a solution such as a sterile aqueous solution,often a saline solution, or they can be provided in powder form. Suchexcipients can be substantially inert, if desired.

In some embodiments, a subject immunogenic composition comprises asubject fHbp present in a vesicle. In some embodiments, a subjectimmunogenic composition comprises a subject fHbp present in an MV. Insome embodiments, a subject immunogenic composition comprises a subjectfHbp present in an OMV. In some embodiments, a subject immunogeniccomposition comprises a mixture of MV and OMV comprising a subject fHbp.Vesicles, such as MV and OMV, are described above.

The antigenic compositions can further contain an adjuvant. Examples ofknown suitable adjuvants that can be used in humans include, but are notnecessarily limited to, alum, aluminum phosphate, aluminum hydroxide,MF59 (4.3% w/v squalene, 0.5% w/v TWEEN 80™, 0.5% w/v SPAN 85),CpG-containing nucleic acid (where the cytosine is unmethylated), QS21,MPL, 3DMPL, extracts from Aquilla, ISCOMS, LT/CT mutants,poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A,interleukins, and the like. For experimental animals, one can useFreund's, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/TWEEN80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against the immunogenicantigen or antigenic epitope thereof.

Further exemplary adjuvants to enhance effectiveness of the compositioninclude, but are not limited to: (1) oil-in-water emulsion formulations(with or without other specific immunostimulating agents such as muramylpeptides (see below) or bacterial cell wall components), such as forexample (a) MF59™ (WO 90/14837; Chapter 10 in Vaccine design: thesubunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995),containing 5% Squalene, 0.5% Tween 80, and 0.5% SPAN 85 (optionallycontaining MTP-PE) formulated into submicron particles using amicrofluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5%PLURONIC-blocked polymer L121, and thr-MDP either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion, and (c) RIBI™ adjuvant system (RAS), (Ribi Immunochem,Hamilton, Mont.) containing 2% Squalene, 0.2% TWEEN 80, and one or morebacterial cell wall components such as monophosphorylipid A (MPL),trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferablyMPL+CWS (Detox™); (2) saponin adjuvants, such as QS21 or Stimulon™(Cambridge Bioscience, Worcester, Mass.) may be used or particlesgenerated therefrom such as ISCOMs (immunostimulating complexes), whichISCOMS may be devoid of additional detergent e.g. WO 00/07621; (3)Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA);(4) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12 (WO99/44636), etc.), interferons (e.g. gamma interferon),macrophage colony stimulating factor (M-CSF), tumor necrosis factor(TNF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL(3dMPL) e.g. GB-2220221, EP-A -0689454, optionally in the substantialabsence of alum when used with pneumococcal saccharides e.g. WO00/56358; (6) combinations of 3dMPL with, for example, QS21 and/oroil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231;(7) oligonucleotides comprising CpG motifs (Krieg Vaccine 2000, 19,618-622; Krieg Curr Opin Mol Ther 2001 3:15-24; Roman et al., Nat. Med,1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Daviset al, J. Immunol, 1998, 160, 810-876; Chu et al., J. Exp. Med, 1997,186, 1623-1631; Lipford et al, Ear. J. Immunol., 1997, 27, 2340-2344;Moldoveami et al., Vaccine, 1988, 16, 1216-1224, Krieg et al., Nature,1995, 374, 546-549; Klinman et al., PNAS USA, 1996, 93, 2879-2883;Ballas et al, J Immunol, 1996, 157, 1840-1845; Cowdery et al, J Immunol,1996, 156, 4570-4575; Halpern et al, Cell Immunol, 1996, 167, 72-78;Yamamoto et al, Jpn. J. Cancer Res., 1988, 79, 866-873; Stacey et al, J.Immunol., 1996, 157, 2116-2122; Messina et al, J. Immunol, 1991, 147,1759-1764; Yi et al, J Immunol, 1996, 157, 4918-4925; Yi et al, J.Immunol, 1996, 157, 5394-5402; Yi et al, J Immunol, 1998, 160,4755-4761; and Yi et al, J Immunol, 1998, 160, 5898-5906; Internationalpatent applications WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100,WO 98/55495, WO 98/37919 and WO 98/52581, i.e. containing at least oneCG dinucleotide, where the cytosine is unmethylated; (8) apolyoxyethylene ether or a polyoxyethylene ester e.g. WO 99/52549; (9) apolyoxyethylene sorbitan ester surfactant in combination with anoctoxynol (WO 01/21207) or a polyoxyethylene alkyl ether or estersurfactant in combination with at least one additional non-ionicsurfactant such as an octoxynol (WO 01/21152); (10) a saponin and animmunostimulatory oligonucleotide (e.g. a CpG oligonucleotide) (WO00/62800); (11) an immunostimulant and a particle of metal salt e.g. WO00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO 99/11241;(13) a saponin (e.g. QS21)+3dMPL+IM2 (optionally+a sterol) e.g. WO98/57659; (14) other substances that act as immunostimulating agents toenhance the efficacy of the composition. Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), etc. Adjuvants suitable foradministration to a human are of particular interest.

The antigen compositions may contain other components, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium,carbonate, and the like. The compositions may contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example, sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate and the like.

The concentration of the subject fHbp in a formulation can vary widely(e.g., from less than about 0.1%, usually at or at least about 2% to asmuch as 20% to 50% or more by weight) and will usually be selectedprimarily based on fluid volumes, viscosities, and patient-based factorsin accordance with the particular mode of administration selected andthe patient's needs.

The fHbp-containing formulations can be provided in the form of asolution, suspension, tablet, pill, capsule, powder, gel, cream, lotion,ointment, aerosol or the like. It is recognized that oral administrationcan require protection of the compositions from digestion. This istypically accomplished either by association of the composition with anagent that renders it resistant to acidic and enzymatic hydrolysis or bypackaging the composition in an appropriately resistant carrier. Meansof protecting from digestion are well known in the art.

The fHbp-containing formulations can also be provided so as to enhanceserum half-life of fHbp following administration. For example, whereisolated fHbps are formulated for injection, the fHbp may be provided ina liposome formulation, prepared as a colloid, or other conventionaltechniques for extending serum half-life. A variety of methods areavailable for preparing liposomes, as described in, e.g., Szoka et al.,Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871,4,501,728 and 4,837,028. The preparations may also be provided incontrolled release or slow-release forms.

Immunization

The present disclosure provides a method of inducing an immune responseto at least one Neisserial strain in a mammalian host. The methodsgenerally involve administering to an individual in need thereof aneffective amount of a subject immunogenic composition.

The fHbp-containing antigenic compositions are generally administered toa human subject that is at risk of acquiring a Neisserial disease so asto prevent or at least partially arrest the development of disease andits complications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for therapeutic usewill depend on, e.g., the antigenic composition, the manner ofadministration, the weight and general state of health of the patient,and the judgment of the prescribing physician. Single or multiple dosesof the antigenic compositions may be administered depending on thedosage and frequency required and tolerated by the patient, and route ofadministration.

The fHbp-containing antigenic compositions are generally administered inan amount effective to elicit an immune response, particularly a humoralimmune response, e.g., a bactericidal antibody response, in the host. Asnoted above, amounts for immunization will vary, and can generally rangefrom about 1 μg to 100 μg per 70 kg patient, usually 5 μg to 50 μg/70kg. Substantially higher dosages (e.g. 10 mg to 100 mg or more) may besuitable in oral, nasal, or topical administration routes. The initialadministration can be followed by booster immunization of the same ofdifferent fHbp-containing antigenic composition. Usually vaccinationinvolves at least one booster, more usually two boosters.

In general immunization can be accomplished by administration by anysuitable route, including administration of the composition orally,nasally, nasopharyngeally, parenterally, enterically, gastrically,topically, transdermally, subcutaneously, intramuscularly, in tablet,solid, powdered, liquid, aerosol form, locally or systemically, with orwithout added excipients. Actual methods for preparing parenterallyadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 15th ed., Mack Publishing Company,Easton, Pa. (1980).

An anti-fHbp immune response can be assessed by known methods (e.g. byobtaining serum from the individual before and after the initialimmunization, and demonstrating a change in the individual's immunestatus, for example an immunoprecipitation assay, or an ELISA, or abactericidal assay, or a Western blot, or flow cytometric assay, or thelike).

The antigenic compositions can be administered to a human subject thatis immunologically naive with respect to Neisseria meningitidis. In aparticular embodiment, the subject is a human child about five years oryounger, and preferably about two years old or younger, and theantigenic compositions are administered at any one or more of thefollowing times: two weeks, one month, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11months, or one year or 15, 18, or 21 months after birth, or at 2, 3, 4,or 5 years of age.

It may be generally desirable to initiate immunization prior to thefirst sign of disease symptoms, or at the first sign of possible oractual exposure to infection or disease (e.g., due to exposure orinfection by Neisseria).

Methods of Screening

In one example, a method of evaluating the efficacy of a subject fHbp ina vaccine composition involves: (a) immunizing a host animal (e.g., anon-human mammalian host animal, such as a rodent, e.g., a mouse) with acomposition comprising a fHbp of the present disclosure; and (b)measuring levels of bactericidal antibodies in the host. The subjectmethod may also include assessing the susceptibility of a host animaladministered with a vaccine comprising a subject fHbp to a bacterialpathogen.

In another example, the method can involve making and identifyingantibodies elicited by the subject fHbp. The method involves isolatingantibodies from the host animal that have binding affinity to the fHbp,contacting a bacterial cell with the isolated antibodies; and assessingbinding of the antibody to the bacterial cell. Additional steps mayinclude assessing the competitive binding of the antibody to fHbp withhuman factor H; assessing the bactericidal activity against a bacterialpathogen when the antibody is administered to an animal contracted withthe bacterial pathogen. In some embodiments, the antibody is in anantibody population, and the method can further comprise: isolating oneor more antibodies of the antibody population that bind the bacterialcell. A featured aspect is isolated antibody that is bactericidalagainst the bacterial cell, which may include, for example,complement-mediated bactericidal activity and/or opsonophagocyticactivity capable of decreasing the viability of the bacteria in humanblood.

Bacterial pathogens of particular interest are N. meningitidis of any orall variant groups, of diverse capsular groups, such as N. meningitidisSerogroup B, N. meningitidis Serogroup C, N. meningitidis Serogroup X,N. meningitidis Serogroup Y, N. meningitidis Serogroup W-135, and thelike.

Methods of Evaluating a Response to a fHbp

The present disclosure provides methods for determining the likelihoodthat a fHbp will elicit a bactericidal response in an individual; andmethods of evaluating a variant fHbp for suitability for inclusion in animmunogenic composition.

Determining the Likelihood that a fHbp Will Elicit a BactericidalResponse

The present disclosure provides a method of determining the likelihoodthat a fHbp (e.g., a fHbp present in a Neisserial vaccine) will elicit abactericidal response in an individual to at least one Neisseriameningitidis strain. The method generally involves determining theability of antibody, present in serum obtained from an individual whohas been immunized with a fHbp, to inhibit binding of fH to fHbp.Inhibition of binding of fH to fHbp by the antibody at a level that isat least about 10% higher, at least about 25%, at least about 50%, atleast about 75%, at least about 2-fold, at least about 10-fold, at leastabout 50-fold, at least about 100-fold, or greater than 100-fold, thanthe level of inhibition of fH to fHbp by a control antibody thatinhibits fH binding to fHbp but that does not generate a bactericidalresponse, indicates that the fHbp is likely to elicit a bactericidalresponse to at least one Neisseria meningitidis strain. In someembodiments, the fHbp is a non-naturally occurring fHbp that has loweraffinity for human factor H (fH) than fHbp ID 1, as described above.

The degree of inhibition of binding of fH to fHbp by antibody elicitedto a fHbp variant can be determined using an assay as described herein,or any other known assay. For example, the fH and/or the fHbp cancomprise a detectable label, and inhibition of fH/fHbp binding can beassessed by detecting the amount of labelled component present in anfH/fHbp complex and/or detecting the amount of label present in free fHand/or free fHbp (e.g., fH or fHbp not in an fH/fHbp complex).

In one example, assays to assess fH binding to an fHbp involve use offHbp immobilized on a support (e.g., fHbp immobilized on the well of amicrotiter plate). A mixture of a fixed concentration of human fH withdilutions of the test antibodies (e.g., antiserum, e.g., from a human ornon-human test animal (e.g., mouse) that has received anantibody-eliciting dosage of an immunogenic composition) are added tothe wells and incubated for an amount of time sufficient to allow forantibody binding. After washing the wells, bound fH is detected with aspecific anti-fH antiserum (e.g., goat or donkey) containing a labeledcomponent, or a secondary labeled antibody (e.g., rabbit anti-goat oranti-donkey anti-serum). Percent inhibition of bound fH can becalculated by the amount of bound fH in the absence of added human ormouse antibody.

In another variation of such assays, binding of fH to live bacteria inthe presence or absence of test antisera is assessed by flow cytometry.Bacterial cells are incubated with a fixed concentration of fH (e.g.,detectably labeled fH) and different dilutions of test sera containingantibody. The bacteria are washed and bound fH is detected (e.g., asdescribed above).

Thus, the ability of antiserum from an individual immunized with a fHbpto inhibit fH/fHbp binding serves as a surrogate for directly assessingbactericidal activity of the antiserum. A method of the presentdisclosure for determining the likelihood that a fHbp will elicit abactericidal response in an individual can provide information to aclinician or other medical personnel as to whether a particularimmunogenic composition has been effective in eliciting a bactericidalresponse in an individual.

Immunized individuals can have a similar serum IgG anti-fHbp antibodytiter by ELISA. Antisera that provides for overall better inhibition offH binding is indicative of a more effective, better quality anti-fHbpantibody response and will confer greater protection. Thus, for example,if in comparing the anti-Neisserial antibody response in two individuals(by the anti-fHbp antibodies, i.e, a serum dilution of 1:10,000 inhibitscompared to a dilution of 1:3000 by the other individual) the individualwith the higher inhibitory activity has better quality anti-fHbpantibody that will confer greater protection. The fH inhibition assay isthus a surrogate for complement-mediated bactericidal titer assays,which complement-mediated bactericidal titer assays are generally moretime consuming and difficult to measure than fH inhibition.

Evaluating a Variant fHbp

The present disclosure provides methods of assessing or predicting thelikelihood that a fHbp variant will be efficacious in eliciting abactericidal antibody response in an individual. The methods generallyinvolve assessing the ability of antibody specific for the fHbp variantto inhibit binding of fH to fHbp. The strength of inhibition of bindingof fH to fHbp by antibody elicited by immunizing with an fHbp variantpositively correlates with bactericidal activity of antibody elicited tothe fHbp variant. A fHbp variant that elicits antibody that inhibitsbinding of fH to fHbp at a high serum dilution is considered a suitablecandidate for a vaccine for eliciting protection against one or morestrains of Neisseria.

For example, the present disclosure provides a method of determining thelikelihood that a non-naturally occurring fHbp that has lower affinityfor human fH than fHbp ID 1 will elicit bactericidal antibodies in anindividual to at least one Neisseria meningitidis strain. The methodgenerally involves determining the ability of an antibody elicited in atest non-human animal to the non-naturally occurring fHbp to inhibitbinding of fH to fHbp. Inhibition of binding of fH to fHbp by theantibody elicited to the non-naturally occurring fHbp at a level that isat least about 10%, at least about 25%, at least about 50%, at leastabout 75%, at least about 2-fold, at least about 10-fold, at least about50-fold, at least about 100-fold, or greater than 100-fold, higher thanthe level of inhibition of fH to fHbp by an antibody elicited in thetest non-human animal to fHbp ID 1 indicates that the non-naturallyoccurring fHbp is likely to elicit a bactericidal response to at leastone Neisseria meningitidis strain.

Suitable test non-human animals include, e.g., mice, rats, rabbits, andthe like. The degree of inhibition of binding of fH to fHbp by antibodyelicited to a fHbp variant can be determined using an assay as describedherein, or any other known assay. Bactericidal activity of an antibodyis readily determined using an assay as described herein, or any otherknown assay.

A subject method for determining the likelihood that a givennon-naturally occurring fHbp that has lower affinity for human fH thanfHbp ID 1 will elicit bactericidal antibodies in an individual to atleast one Neisseria meningitidis strain is useful for identifyingsuitable immunogens (and/or eliminating unsuitable immunogens), e.g., inthe course of vaccine development.

EXAMPLES

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Overview of Examples

Factor H (fH) is present in high concentrations in serum (˜200 to 800μg/ml). Binding of fH to fHbp is specific for human fH (Granoff et al.(2009) Infect Immun 77:764). One implication is that when humans areimmunized with fHbp, the molecule can form a complex with fH. Incontrast, when non-human primates or other experimental animals areimmunized, the antigen is presented to the immune system without boundfH. In humans, the presence of fH in a complex with fHbp may affect theimmunogenicity of fHbp (e.g. by covering epitopes and affecting antigenpresentation).

Provided herein is evidence that the presence of human fH decreasesprotective antibody responses to a fHbp vaccine that binds fH. Further,while certain mutant vaccines with one or two amino acid substitutionsdo not bind fH (e.g., E218A and/or E239A), the specific mutations usedto alter the molecule caused changes that decreased the ability of thevaccines to elicit serum bactericidal antibodies. Additional singleamino acid mutants (e.g. R41S or R41A mutants of fHbp ID 1; R41S mutantsof fHbp ID 4, 9, 74 or chimeric fHbp I; R130A of fHbp ID 1; R80A, D211A,E218A, E248A, or G236I mutants of fHbp ID 22; a T221A/H223A mutant offHbp ID 22; R41S/K113A, R41S/K119A, R41S/D121A, or R41S/K113A/D121Amutants of fHbp ID 77; and a K199A or E218A mutant of fHbp ID 28) wereidentified that had decreased fH binding. A fHbp vaccine with the R41Smutation did not have decreased ability to elicit bactericidalantibodies and in the presence of human fH gave higher protectiveantibody responses than the wildtype fHbp ID 1 vaccine that bound fH.Other mutations such as K241E of fHbp ID 1 or E241K in fHbp ID 15, whichfrom the crystal structure of fHbp ID 1 are predicted to be in contactwith fH, had no effect on fH binding. Further the R41S mutation, whichdecreased fH binding in fHbp ID 1, 4, 9, and 74, did not decrease fHbinding in fHbp ID 22 or 77. Mutations (such as R41S in fHbp ID 1 andother mutations discussed below) that decrease fH binding but haveminimal or no effect on the conformation of fHbp such that the mutantvaccine elicits bactericidal antibodies can result in superior vaccinecandidates. Thus, fHbp variants are provided that maintain and present aconformational epitope bound by bacteridal antibodies that havebactericidal activity toward one or more Neisseria meningitidis strains.

Details of the studies that led to this discovery are set out below.

Materials and Methods

Human fH Transgenic Mouse Model.

The 3.9 kbp human complement fH (cfh) cDNA was cloned into plasmidpCAGGS (Niwa et al. (1991) Gene 108:193-9). BALB/c mouse embryos weremicroinjected with the ˜6 kbp SalI-PstI restriction fragment, andimplanted into pseudo-pregnant female BALB/c mice. Expression of humanfH in sera of pups was detected by Western blotting.

Serum Human fH Concentrations.

To distinguish human and mouse fH, a fHbp capture enzyme-linkedimmunosorbent assay (ELISA) that specifically binds human fH was used.Recombinant fHbp (2 μg/ml) in sterile phosphate buffered saline (PBS)was added to the wells of microtiter plates. After blocking with 1%bovine serum albumin (BSA), dilutions of pre-immune mouse or human serawere added. Bound human fH was detected using sheep anti-human fHantiserum (Lifespan Biosciences, Seattle, Wash.; 1:2000 dilution). TheELISA was developed with anti-sheep IgG conjugated to alkalinephosphatase. The phosphatase substrate p-nitrophenyl phosphate(Sigma-Aldrich, St. Louis, Mo.) was added and after incubation at roomtemperature for 30 min, the optical density at 405 nm was measured. fHconcentrations were determined in comparison to dilutions of a humanreference serum containing 471 μg/ml of fH. As a control, fH wasmeasured in 25 sera from adult subjects in the San Francisco Bay areawho participated in an IRB-approved protocol to screen normal sera ascomplement sources for serum bactericidal assays.

Recombinant fHbp Vaccines.

Recombinant fHbp wild-type and R41S mutant proteins were purified asdescribed (Beernink P T et al. (2008) Infect Immun 76:2568-2575).Vaccine immunogenicity was evaluated in six- to eight-week old BALB/cwild-type or human fH transgenic mice, using a protocol approved by theUniversity of Massachusetts Medical School Institutional Animal Care andUse Committee. Three doses of vaccine containing 20 μg of fHbp adsorbedwith 600 μg of aluminum hydroxide were administered intraperitoneally atthree-week intervals. The control meningococcal group C conjugatevaccine (Meningitec; Wyeth, Montreal, Canada) contained 2 μg ofpolysaccharide and 3 μg of CRM₁₉₇ adsorbed with 100 μg of aluminumphosphate.

Statistical Analyses.

Two-tailed Student's t tests were used to compare reciprocal geometricmean titers (GMT) of serum antibody responses between two independentgroups of mice. A one-tailed t test was also used to examine whetheranti-fHbp antibody responses of transgenic mice immunized with thewild-type fHbp vaccine were not lower than immunized wild-type mice.General linear regression models were used to test whether the type offHbp vaccine and human fH concentrations affected serum bactericidalantibody responses. To meet normality assumption, both serumbactericidal antibody measurements and fH concentrations were logintransformed in regression and correlation analyses. A two-tailed P-valueof less than or equal to 0.05 was considered statistically significant.

Example 1: Binding of Human Fh Decreases the Immunogenicity of a fHbpVaccine

Binding of fH to fHbp may cover epitopes and impair antibody responsesdirected at portions of the fHbp molecule exposed on the surface of thebacteria, which are most effective for bactericidal activity. Sincebinding of human fH to fHbp is specific for human fH, the effect of fHon vaccine immunogenicity was investigated using a human fH transgenicanimal model. The human fH concentrations in sera were measured from thetransgenic mice using a fHbp capture ELISA described above that isspecific for human fH. Control wells contained a purified human fH atconcentrations ranging from 0.15 to 5 μg/ml (FIG. 1, panel A).Experimental wells contained different dilutions of transgenic mousesera (serial 2-fold dilutions starting at 1:100). The human fHconcentrations in sera from the transgenic mice were >100 μg/ml. Theserum factor H-negative littermates had concentrations <12 μg/ml, whichwas the lower limit of the assay). Known wild-type mice also had humanfH<12 μg/ml). For comparison, fH concentrations in stored serum samplesfrom adult complement donors >100 μg/ml) (FIG. 1, panel B). In theexperiments described below, littermates of transgenic mice with <12μg/ml or known wildtype mice will be referred to as “wildtype” mice.

In Study 1, human fH transgenic or wild-type mice were immunized with arecombinant fHbp vaccine that bound human fH (Table 1 below). Threeweeks after the third injection of vaccine, the serum bactericidalantibody responses of the transgenic mice were 8-fold lower than thewild-type mice whose serum fH did not bind the vaccine (reciprocal GMTof 59 vs. 453 in wild-type mice, P=0.03). Study 1 did not include acontrol vaccine that did not bind fH. Therefore, it should be determinedwhether the lower immunogenicity of the fHbp vaccine in the transgenicmice resulted from binding of the vaccine antigen with human fH, orwhether the mice might have had lower serum antibody responses tovaccine antigens in general. In Study 2, the fHbp vaccination wasrepeated and included groups of transgenic and wild-type mice immunizedwith a control meningococcal group C polysaccharide-CRM₁₉₇ conjugatevaccine that did not bind human fH. The respective serum IgG andbactericidal antibody responses of the transgenic mice immunized withthe meningococcal conjugate vaccine were not significantly differentfrom those of the wild-type mice (FIG. 2). As observed in Study 1,transgenic mice immunized in Study 2 with the fHbp vaccine that boundhuman fH had lower serum bactericidal antibody responses (reciprocal GMTof 31 vs. 115 in wild-type mice, P=0.05, one tailed T test). Further,there was an inverse correlation between the human fH concentrations inthe sera of the transgenic mice and serum bactericidal antibodyresponses to the fHbp vaccine that bound human fH (FIG. 3, panel A;Pearson correlation coefficient, r=−0.65; P=0.02). Thus, the higher theserum human fH concentration, the lower the serum bactericidal responseto the vaccine

In both studies, the serum IgG anti-fHbp antibody responses of thetransgenic mice were lower than the wild-type mice (study 1, reciprocalGMT of 30,000 vs. 97,000, P=0.03; study 2, reciprocal GMT of 107,000 vs.190,000 (P=0.025). Collectively the data indicated that binding of humanfH to the fHbp vaccine impaired both IgG anti-fHbp antibody titers andbactericidal antibody responses.

TABLE 1 Complement-mediated serum bactericidal antibody responses ofwild-type or human fH transgenic mice immunized with a recombinant fHbpvaccine that bound human fH 1/Bactericidal Titer Geo. Study Mice No.Mice fHbp Vaccine Mean Log₁₀ ± SE Mean 1 WT 7 WT 2.66 ± 0.21^(a) 453 1fH Tg 10 WT 1.77 ± 0.27^(b) 59 2 WT 14 WT 2.06 ± 0.20^(c) 115 2 fH Tg 14WT 1.49 ± 0.27^(d) 31

The WT fHbp vaccine bound fH. ^(a,b)P=0.03 (two tailed); ^(c,d)P=0.05(one tailed hypothesis based on the results from study 1.

Example 2: fHbp Mutants at Positions 218 and/or 239 Result in DecreasedBinding to fH

A fHbp mutant with two alanine substitutions at glutamate residues 219and 239 (E218 and E239) was found to eliminate fH binding (Schneider M Cet al. (2009) Nature 458:890-3). Recombinant fHbp mutants E218A, E239Aand E218A/E239A were prepared by purification via Ni²⁺ affinitychromatography as described (Beernink et al (2010) Clin Vaccine Immunol17:1074-8). Binding of human fH to the fHbp mutants was performed byELISA using purified recombinant mutant or WT fHbp as the antigen on theplate as described above. Using this method, it was confirmed that thedouble mutant had decreased binding of fH (FIG. 4). Further, the fHbpmutants with individual mutations at E218 or E239 also had decreasedbinding of human fH (FIG. 4).

Example 3: The E218a and/or E239A Mutant fHbps have ImpairedImmunogenicity in Wild-Type Mice

The respective wild-type ID1 fHbp and E218A/E239A double mutant fHbpwere subjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). Recombinant fHbps expressed in Escherichiacoli were purified by Ni²⁺ affinity chromatography and five μg ofpurified protein were loaded onto the gel. A NuPAGE 4-12% polyacrylamidegradient gel (Invitrogen, Carlsbad, Calif.) was run at 200 V for 45 minin MES buffer (Invitrogen) and stained with Simply Blue Safe Stain(Invitrogen). The molecular mass standards were Kaleidoscope Broad Range(Bio-Rad, Richmond, Calif.). The proteins were visualized by COOMASSIEBLUE staining and had similar masses and purity (FIG. 5).

The inhibition ELISA (Beernink et al (2010) Clin Vaccine Immunol17:1074-8) was performed for both wild-type fHbp ID1 and the E218A/E239Adouble mutant. The ELISA plate was coated with recombinant fHbp (ID 1)at 2 μg/ml overnight at 4° C. After blocking with PBS/1% BSA, murineanti-fHbp MAbs (JAR 1 or JAR 4 at 0.5 μg/ml; mAb 502 or JAR 5 at 0.1μg/ml) and serial five-fold dilutions of soluble recombinant proteininhibitor starting at 50 μg/ml (final concentrations) were pre-mixed andadded to the wells of the plate and incubated for 1 h at 37° C. Bindingof the MAbs was detected with alkaline phosphatase conjugated goatanti-mouse IgG (1:10,000; Sigma-Aldrich) for 1 h at room temperature.The plate was developed as described above in Example 1. The resultsshow that the epitopes recognized by four anti-fHbp MAb were preservedin the E218A/E239A double mutant compared with wild-type fHbp as judgedby the ability of the soluble mutant or wildtype protein to inhibitbinding of each of the mAbs to wildtype fHbp, which was adsorbed to thewells of the microtiter plate (FIG. 6).

The thermal stability of the fHbps was also determined. Purifiedrecombinant proteins were dialyzed against PBS (Roche Applied Science,Indianapolis, Ind.) overnight at 4° C. The concentration was measured byUV absorbance at 280 nm using a molar extinction coefficient of 0.8940M⁻¹cm⁻¹ and adjusted to a concentration of 0.5 mg/ml. Degassed proteinsolution and PBS were loaded into the sample and reference cells,respectively, of a VP-DSC microcalorimeter (MicroCal, Northampton,Mass.). The heating rate was 60° C./h and the middle gain setting wasused. The data were baseline corrected using a buffer vs. buffer scanand normalized based on the protein concentration. The E218A/E239Adouble mutant had similar thermal stability as that of the wild-typeprotein (FIG. 7A), as did an R41S mutant (FIG. 7B).

The immunogenicity of the fHbp ID 1 wildtype (WT) and mutant E218A/E239Avaccines was measured in four studies in mice (Studies 3-6). In Study 3,CD-1 mice were immunized with three doses of recombinant WT or mutantfHbp adsorbed with Freund's Adjuvant (FA) or aluminum hydroxide(Al(OH)₃); in Study 4, CD-1 mice were immunized with one dose ofwild-type (WT) or mutant fHbp adsorbed with aluminum hydroxide(Al(OH)₃); in studies 5 and 6, BALB/c mice were immunized with threedoses of WT or mutant fHbp adsorbed with aluminum hydroxide (Al(OH)₃).In Study 3, the sera were pooled (3 pools per vaccine group, each poolfrom sera of 3 to 4 mice). In Studies 4, 5 and 6, individual sera wereassayed (N=7 to 9 mice per vaccine group).

To measure serum anti-fHbp IgG titers, the ELISA plates were sensitizedwith recombinant fHbp ID 1 at 2 μg/ml overnight at 4° C. After blockingwith PBS/1% BSA, mouse antiserum dilutions (serial five-fold starting at1:100) were added to the wells of the plate and the plate was incubatedfor 1 h at 37° C. The bound anti-fHbp antibodies were detected with goatanti-mouse IgG (1:10,000; Sigma-Aldrich, St. Louis, Mo.) for 1 h at roomtemperature. The plate was developed using p-nitrophenyl phosphatesubstrate (Sigma-Aldrich, St. Louis, Mo.) at room temperature for 30 minand the optical density at 405 nm was measured.

As shown in FIGS. 7C and D, in all four studies (Studies 3, 4 and 5,FIG. 7C; and study 6, FIG. 7D), the E218A/E239A double mutant haddecreased serum IgG anti-fHbp antibody responses in conventional BALB/cor CD-1 mice compared with the control wildtype fHbp vaccine. Therespective differences were significant (P<0.05) for studies 4, 5 and 6when individual sera were assayed instead of the pooled sera used instudy 3. Study 6 (FIG. 7D) also included a mutant fHbp vaccine with asingle amino acid substitution, E239A, which showed lower IgG titersthan the WT fHbp vaccine (p=0.05).

Table 2, below, summarizes the serum bactericidal antibody responses tothe mutant E218A/E239A vaccine as measured against group B strainH44/76. In all of the studies, the mutant vaccine elicited lower serumbactericidal titers. The respective differences were significant instudies 4, 5 and 6 (P<0.05).

In these studies, neither the double mutant nor wildtype fHbp boundmouse fH. Nevertheless, the lower immunogenicity of the E218A/E239Amutant indicated that epitopes important for eliciting protectivebactericidal antibodies were perturbed by introduction of the twomutations. As such, the E218A/E239A mutations that eliminated fH bindingdid not necessarily maintain optimal structural vaccine characteristicsfor eliciting protective antibodies.

TABLE 2 Complement-mediated serum bactericidal antibody responses ofwild- type mice immunized with the E218A/E239A mutant fHbp vaccine. fHbpVaccine WT E218A/E239A Mutant Mouse No. 1/Mean Log₁₀ 1/Mean Log₁₀Study^(a) Strain Doses Titer ± 2SE 1/GMT Titer ± 2SE 1/GMT 3 CD-1 3 2.63± 0.54  427 2.31 ± 0.25  206 4 CD-1 1 1.26 ± 0.36^(c) 18 0.80 ± 0.14^(c)6 5 BALB/c 3 3.30 ± 0.30^(d) 1986 1.68 ± 0.60^(d) 48 6 BALB/c 3 1.89 ±0.22^(e) 77 0.95 ± 0.26^(e) 9 Bactericidal activity was measured withhuman complement against strain H44/76, In studies 3, 5 and 6, the micewere immunized with three doses of vaccine. In study 4, one dose wasgiven. ^(c)P < 0.05 ^(d)P < 0.05 ^(e)P = 0.01

Example 4: Identification of a Natural fHbp Variant with Decreased fHBinding

In studies of fH binding by naturally-occurring fHbp variants within thepreviously described sub-family B (Fletcher et al (2004) Infect Immun72:2088-2100), also referred to as variant 1 group (Masignani et al.(2003) supra), recombinant proteins of two fHbp variants, IDs 14 and 15,showed significantly less concentration-dependent fH binding than thatof fHbp protein ID 1 (FIG. 8). In contrast, fHbp ID 14 showed theexpected concentration-dependent binding with anti-fHbp MAbs JAR 4 andJAR 5, and fHbp ID 15 showed the expected binding with anti-fHbp mAb JAR5 but not JAR 4 (Note, fHbp ID 15 was not expected to bind with JAR 4because this protein lacks the epitope) (Beernink et al. (2009) MolImmunol 46:1647-1653; Pajon et al. (2009) Vaccine 28:2122-2129)).

Previous data indicated that fHbp representative of variant groups 1, 2or 3 showed similar respective binding with fH (Shaughnessy J et al.(2009) Infect Immun 77:2094-103). As such, the decreased binding of fHby two naturally-occurring fHbp variants (fHbp IDs 14 and 15 as shown inFIG. 8) was unexpected.

The data obtained as represented in FIG. 8 showing low fH binding by twonaturally-occurring fHbp variants indicates that amino residuescontributing to such lower fH binding can be identified by analysis ofalignments of the fHbp amino acid sequences of high- and low-fH binders.This strategy would be different from one that targets conservedresidues, such as the E218A and E239A residues.

fHbp variants can be subclassified according to different combinationsof five variable segments, each derived from one of two geneticlineages, designated α- or β-types (Pajon et al. (2009) Vaccine 28:2122;Beernink and Granoff (2009) Microbiology 155:2873-83). fHbp ID 1 withhigh fH binding and ID 14 with low fH binding are both in modular groupI (all five segments are alpha-types). In contrast, the second low fHbinder, fHbp ID 15, is in modular group IV, which are natural chimeras(with a β-type A segment and α-type B, C, D and E segments). Therefore,as a control for the β A segment of peptide ID 15, the sequence of thenaturally high fH binding variant peptide ID 28 was used, which containsonly β segments (modular group II). The respective amino acid alignmentsare shown in FIGS. 19A and 19D. For purposes of comparison of thesequences of the different variants, specific residues are referredherein based on the numbering of fHbp ID 1. One of these amino acidresidues, serine (S), at position 41 of the A (β) segment of peptide 15(low fH binding) differed from the proline (P) residue of the control Aβ segment of peptide 28 (high fH binding). A second amino acid, E atposition 241 in the E α segments of both low fH binding variants,differed from that of K at position 241 of the high fH binding variantpeptide 1.

Example 5: Identification of New fHbp Mutants at Position 41 withDecreased fH Binding

The arginine residue at position 41 (R41) formed a charged hydrogen-bondwith fH (FIG. 9, panel A). Arginine was replaced by serine to eliminatethis charged bond (S41, lower right inset panel). Wells of microtiterplates were coated with recombinant WT fHbp ID 1 or the R41S mutantID 1. By ELISA, the R41S mutant did not bind human fH (FIG. 10, panelA). Control anti-fHbp MAbs, JAR 4 and JAR 5, bound almost identically toboth the mutant fHbps and wild-type fHbp (FIG. 10, panels B and C).These controls indicated that comparable amounts of the respectiveproteins were adsorbed to the wells of the microtiter plate. Further,the R41S mutation, which was in the same domain and in close proximityto the fHbp conformational epitope recognized by the JAR 4 MAb (BeerninkP T (2009) Mol Immunol 46:1647-53), did not affect binding of the MAb.An additional mutation in fHbp ID 1 in which alanine was substituted forarginine, R41A also did not bind fH (FIG. 10A). Thus substitutions otherthan serine at position 41 also can decrease fH binding.

In surface plasmon resonance experiments, human fH (2400 response units)was immobilized on a CMS chip (GE Healthcare, Piscataway, N.J.) viaamine coupling and binding of soluble fHbp was measured. The R41S mutantprotein (0.5 μM) showed no binding with fH (−0.6 response units)compared with +22.5 response units with 0.5 μM of the respectivewild-type fHbp antigen, which independently confirmed the ELISA results.The R41S mutant protein also had thermal stability compared with that ofthe wild-type fHbp (FIG. 7, panel B).

The R41S mutation also eliminated fH binding when the mutation wasintroduced in other fHbp sequence variants in the variant group 1,modular group I. These included fHbp ID 4, 9, and 74 (FIG. 11, panels A,C, and E, respectively). However, the R41S mutation in three sequencevariants in the variant group 2 (modular groups III or VI) did notdecrease fH binding. These included fHbp ID 19, 22 and 77 (FIG. 12,panels A, C, and E, respectively).

Example 6: Immunogenicity of R41S Mutant fHbp in Wild-Type Mice

In wild-type mice, the R41S mutant fHbp (ID 1) vaccine elicited similarserum bactericidal antibody responses as the wild-type vaccine (Table 3,below, Studies 2 and 6). In study 6, a double mutant fHbp vaccine,E218A/E239A, which previously was reported not to bind to fH (SchneiderM C et al. (2009) Nature 458:890-3), but had impaired immunogenicity inWT mice (Beernink et al. (2010) Clin Vaccine Immunol 17:1074), served asa negative control. This vaccine elicited significantly lowerbactericidal titers (Table 3, Study 6), and thus confirmed the datadescribed in Table 2 above, showing diminished antibody responses to theE218A/E239A vaccine from possible loss of epitopes or minordestabilization of the mutant molecule (Beernink et al. (2010) ClinVaccine Immunol 17:1074-8). In contrast, the normal antibody responsesto the R41S mutant fHbp vaccine indicated that substitution of serinefor arginine did not decrease immunogenicity in a mouse model where fHdid not bind to the mutant or wild-type fHbp vaccines.

TABLE 3 Complement-mediated serum bactericidal antibody responses ofwild-type mice immunized with fHbp recombinant fHbp vaccines.1/Bactericidal Titer Geo. Study Mice No. Mice fHbp Vaccine Mean Log₁₀ ±SE Mean 2 WT 14 WT 2.06 ± 0.20 115^(a) 2 WT 13 R41S 1.92 ± 0.20  83^(b)6 WT 9 WT 1.89 ± 0.22  77^(c) 6 WT 9 E218A/E239A 0.95 ± 0.26  9^(d) 6 WT9 R41S 1.85 ± 0.31  71^(i)

The WT fHbp vaccine bound human fH; the R41S mutant and previouslydescribed E218A/E239 mutant (Schneider et al. (2009) Nature 458:890-3)did not bind human fH. In WT mice, native fH does not bind to eithervaccine (FIG. 1, Panel D): ^(a,b)P=0.62; ^(c,d)P=0.01; ^(d,i)P=0.92, byT tests (two tailed).

Example 7: Serum Bactericidal Antibody Responses of Transgenic MiceImmunized with the R41S Mutant fHbp Vaccine

Human fH transgenic mice immunized with the R41S mutant vaccine that didnot bind human fH had ˜3-fold higher serum bactericidal antibodyresponses than human fH transgenic mice immunized with the controlwildtype fHbp vaccine that bound fH (Study 2, Table 4 below). When thedata were stratified by serum human fH concentrations, mice with fHconcentrations <250 μg/ml showed similar responses to the mutant andwildtype vaccines. However, among mice with human fH concentrations >250μg/ml, those immunized with the R41S mutant vaccine had 10-fold higherbactericidal antibody responses than those immunized with the wildtypefHbp vaccine that bound human fH (P<0.05; Table 4).

TABLE 4 Serum bactericidal antibody responses of human fH transgenicmice immunized with the R41S mutant vaccine 1/Bactericidal Titer HumanMean Geo. Study fH, μg/ml No. Mice fHbp Vaccine Log₁₀ ± SE Mean 2 >10014 WT 1.49 ± 0.27^(a) 31 2 >100 13 R41S 1.98 ± 0.23^(b) 96 Stratifiedanalysis by human fH concentration 2 <250 7 WT 2.02 ± 1.16^(c) 105 2<250 8 R41S 2.11 ± 0.78^(d) 129 2 ≥250 6 WT 0.80 ± 0.10^(e) 6 2 ≥250 5R41S 1.78 ± 0.44^(f) 60

The WT fHbp vaccine bound human fH; the R41S mutant did not bind humanfH. The data for the fH transgenic mice immunized with the wild-typevaccine are shown in study 2, Table 1 above. ^(a,b)Using general linearregression models, the effect of fHbp R41S mutant or wild-type vaccinetype differed by serum human fH concentration on bactericidal titer,P=0.018. ^(c,d)p>0.5. ^(e,f)P=0.05.

In immunized human fH transgenic mice, there was no significantcorrelation between the serum bactericidal antibody responses to themutant fHbp vaccine that did not bind human fH and serum human fHconcentrations (FIG. 3, panel B; r=+0.17; P=0.58), whereas as describedabove (FIG. 3, panel A), in the transgenic mice there was an inversecorrelation with the bactericidal titers elicited by the wild-typevaccine that bound fH (r=−0.65; P=0.02). The respective correlationcoefficients for the two vaccines were significantly different from eachother (P=0.03).

General linear regression models were used to confirm if the type offHbp vaccine (fHbp wild-type or R41S mutant) or the serum human fHconcentration affected the serum bactericidal antibody responses of thetransgenic mice. There was a significant interaction between the type offHbp vaccine and the human fH concentration on the bactericidal response(likelihood ratio test, P=0.018). Based on the regression model, ratiosof the reciprocal serum bactericidal GMTs were estimated for transgenicmice immunized with the R41S mutant vaccine over those of transgenicmice immunized with the fHbp vaccine that bound human fH at variousserum human fH concentrations (FIG. 3, panel C). While there were nosignificant differences in bactericidal responses when serum human fHconcentrations were low (<250 μg/ml), the bactericidal responses to theR41S mutant vaccine were significantly higher when the serum fHconcentrations were higher (fH>250 μg/ml, P<0.05; fH>316 μg/ml, P<0.01).Since many humans have fH concentrations in this range (FIG. 1, panelB), the results in the transgenic mice suggest that mutant fHbpmolecules that do not bind fH can be superior vaccines in humans.

The experimental protocols of various immunization studies presentedabove as well as the results are summarized in the table below.

TABLE 5 Summary of immunization studies in human fH transgenic mice. fHbinding BALB/c Meningococcal to Study mouse strain Vaccine(s) vaccineResults 1 Human fH Tg Factor H binding Yes¹ Lower serum IgG andbactericidal protein (fHbp) antibody response of Tg mice whose Wild-typefHbp No² human fH bound to the vaccine control antigen 2A Human fH TgfHbp Yes¹ Confirmed lower serum IgG and Wild-type fHbp No² bactericidalantibody responses of Tg control mice 2B Human fH Tg Group C PS- No³Wild-type and Tg mice showed CRM conjugate nearly identical respectiveserum IgG Wild-type Group C PS- No⁴ and bactericidal responses to acontrol CRM conjugate control meningococcal vaccine that didn't bind fH2C Human fH Tg fHbp Yes¹ Higher serum bactericidal antibody Human fH TgfHbp R41S No⁵ responses to the mutant fHbp that did mutant not bind fH,especially for the mice with high serum human fH levels For the vaccinethat bound human fH, inverse correlation between serum bactericidaltiter and serum human fH concentration For the mutant vaccine thatdidn't bind human fH, no significant correlation between serumbactericidal titers and serum human fH concentrations Hence, in micevaccinated with mutant fHbp, serum bactericidal titers were independentof the serum human fH concentration.

FIG. 2, panel D provides a schematic illustration of each experimentalprotocol corresponding to the various studies presented herein. Thenumber above each illustration in FIG. 2D corresponds to thesuperscripts in the table above. Group C PS-CRM conjugates are aconjugate of meningococcal group C polysaccharide (PS) and across-reactive mutant diphtheria toxoid (CRM) and are referred to asMenC-CRM in FIG. 2, panel D.

Example 8: The Antibody Repertoire of Transgenic Mice Immunized with theR41S Mutant fHbp Vaccine Preferentially Binds Epitopes Near the fHBinding Site

The importance of binding of human fH that covers fHbp epitopes ineliciting antibodies with protective functional activity was tested. Theability of endogenous human fH present in 1:100 dilutions of sera fromtransgenic mice to bind to fHbp by ELISA was measured. As expected, inthe absence of serum anti-fHbp antibodies, there was similar binding ofhuman fH in pre-immunization sera from the two vaccine groups and thecontrol transgenic (Tg) mice given aluminum hydroxide alone (FIG. 13,panel A). There was no binding in the control WT mice given aluminumhydroxide since the native fH did not bind to fHbp. After vaccination,there was less “free” human fH detected in the sera from mice immunizedwith the R41S mutant fHbp than in the sera from mice immunized with thevaccine that bound human fH (P=0.001, FIG. 13, panel B), or in sera fromtransgenic mice given aluminum hydroxide alone (FIG. 13, panel B). Sincethe respective IgG anti-fHbp antibody titers were similar in the twofHbp vaccine groups FIG. 13, panel D), the lower detectable human fHconcentrations in the R41S post-immunization sera were consistent withgreater ability of the anti-fHbp antibodies to inhibit binding of humanfH to fHbp than the anti-fHbp antibodies elicited by the wildtypevaccine that bound human fH. Individual mouse sera (N=11 per group) werealso tested at different dilutions in the presence of 5% normal humanserum as a source of fH. At 1:100 and 1:400 dilutions, inhibition wassignificantly greater in the R41S mutant vaccine group (P<0.03), FIG.13, panel C). Collectively, the greater fH inhibition in the R41S mutantvaccine group suggested that there were differences in antibodyrepertoire elicited by the two vaccines. For example, antibodieselicited by the mutant fHbp vaccine may have been directed more atepitopes near the fH binding site, which would be more effective inblocking fH binding than the antibody repertoire elicited by the vaccinethat bound fH. Further, antibodies directed at surface-exposed regionsof fHbp that also bind to fH would be expected to have greaterfunctional bactericidal activity.

A significant correlation (Spearman r value, 0.69 and P value of 0.0004)was also observed between the ability of individual mouse sera toinhibit binding of human fH to fHbp and the reciprocal serumbactericidal titer (FIG. 13, panel E). In the serum bactericidalreaction, a decrease in binding of the complement inhibitor fH to thebacterial surface of the test organism may have contributed to thehigher bactericidal titers elicited by the mutant fHbp vaccine. Thus,the ability of the anti-fHbp antibodies to inhibit fH binding predictedprotective antibody activity, which was greater for the R41S vaccine.

Example 9: Identification of Additional Mutants in fHbp ID 1 withDecreased fH Binding

Position 241 is in the fH binding interface of fHbp ID 1. The effect ofamino acid substitutions on binding of fH was investigated at residue241 in the fHbp ID 1 sequence. As shown in FIG. 14, panel A, replacementof residue lysine (K) 241 with glutamate (E) (K241E) in fHbp ID 1 had noeffect on fH binding. The converse substitution, the E241K mutant offHbp ID 15 in modular group IV (FIG. 14, panel C) also showed nosignificant effect on fH binding relative to the wildtype fHbp (<2-fold;FIG. 14, panel C). (Numbering of amino acid residues is based on thesequence of fHbp ID 1.)

In fHbp ID 1, mutations at positions R41, H119, R130, and K241. ThefHbps mutants were produced as described above. The R41A, H119A, R130A,and K241E single substitution mutants were then assessed for binding tohuman fH, and for binding to MAbs.

As shown in FIG. 10, panel A, the R41S substitution and the R41Asubstitution in fHbp ID 1 reduced binding to human fH. As shown in FIG.10, panel B and C, the R41S and the R41A mutants retained binding toMAbs JAR 4 and JAR 5, respectively, which indicated that these epitopesare preserved in the R41S and the R41A mutants.

As shown in FIG. 15, panel A, the H119A and the R130A substitutions infHbp ID 1 reduced binding to human fH. As shown in FIG. 15, the H119Aand the R130A mutants retained binding to MAb JAR5 (panel B) and loweredbinding to MAb JAR4, compared to the corresponding wildtype fHbp ID 1(panel C). These data indicate that the JAR5 epitope is preserved in theH119A and the R130A mutants; and that the JAR 4 epitope is partiallypreserved by the amino acid substitutions.

Example 10: Mutants in fHbp Sequence Variants from fHbp Modular Group IV

The “V_(A)” segments in variant group 1, fHbp sequence variantsclassified as variant 1, modular group IV (FIG. 16) are derived from adifferent genetic lineage (β) than the corresponding “V_(A)” segments invariant 1, modular group I fHbp sequence variants, which are designatedas α segments (Beernink et al (2009) Microbiology 155:2873). Therespective α and β lineages can also be designated as lineages 1 and 2,according to the nomenclature adopted by the pubmlst.org/neisseria/fHbp/website.

In modular group IV fHbp amino acid sequence variants, there often is aserine at position 41 instead of arginine. Substituting proline forserine (S41P) in a mutant of fHbp ID 15 (modular group IV) eliminatedbinding of fH (FIG. 17). Control proteins included recombinant fHbp IDs1 and 28 (naturally high fH binders) and fHbp ID 15 (naturally low fHbinder). Human factor H or anti-fHbp MAb binding to fHbp was measured byELISA as described above. Anti-fHbp MAb JAR 5 showed similar bindingwith WT fHbp IDs 1 and 15, and the S41P mutant of fHbp ID 15 (FIG. 17,panel B). JAR 31 showed the expected binding of fHbp ID 28 (FIG. 17,panel C).

Example 11: R41S Amino Acid Substitutions in fHbp Sequence Variants fromModular Groups III and VI do not Affect fH Binding

All fHbp sequence variants classified as variant 2 are natural chimerasthat contain segments derived from both α and β lineages (FIG. 16).Specifically, the “V_(A)” segments in variant 2 proteins are derivedfrom α lineages and as in modular group I frequently contain an arginineat residue 41 (numbering of the residues according to fHbp ID 1).Although the R41S substitution in all modular group I proteins testedeliminated fH binding (FIGS. 11 and 10 and Table 6), the R41S mutationin fHbp ID 19, 22 and 77 from variant 2 group (modular groups III or VI)did not eliminate fH binding (FIG. 12, panels A, C and E, and Table 7).

Example 12. Synthetic fHbp Chimeric Proteins that do not Bind Human fH

A fHbp chimera I (Beernink and Granoff (2008) Infect. Immun. 76:2568-75)is shown as the last modular schematic in FIG. 16. The junction point atwhich part of fHbp ID 1 (variant 1, modular group I) is fused to part offHbp 77 (variant 2, modular group VI) is G136, which resides in segmentV_(C). In FIG. 16, V_(C) is depicted as half gray and half white in thechimeric protein to represent the fusion of a α lineage sequence to a βlineage sequence in that segment. When the R41S substitution wasintroduced into variant 2 fHbp protein, there was no effect on fHbinding (FIG. 12, panels A, C, and E). In contrast, when the R41Ssubstitution was inserted in the fHbp chimera I protein, the mutationeliminated fH binding. (FIG. 18). This results was not anticipated sincethe only amino acid differences between the respective V_(A) segments ofchimera I and fHbp ID 77 was one amino acid residue (Gly30 in thechimeric antigen instead of Ser30; FIG. 19). In the V_(C) segment, therewere differences in eight of the residues between positions 98 and 135(FIG. 19), which may explain why the R41S mutation eliminated fH bindingin the chimeric protein but not in the natural variant 2 proteins (shownschematically in FIG. 16; and complete amino acid sequence shown in FIG.19A). These observations implicate residues in this portion of the V_(C)region as being important for stability of the fHbp-fH complex in fHbpsin variant 2 group.

Example 13. Effect of Additional Amino Acid Substitutions in fHbp ID 77(Modular Group VI) on Binding of fH

Alanine mutations at positions K113, K119, D121, were introduced intofHbp ID 77 (Modular Group VI, antigenic variant group 2). As notedabove, the residue position number is based on fHbp ID 1. fHbps wereproduced as described above in Materials and Methods.

The ability of these mutants to bind to human fH were tested by ELISA asdescribed above in Example 2 and compared to the corresponding wild-typefHbp. Introducing the K119A mutation increased fH binding approximately4-fold compared to wildtype fHbp ID 77 (FIG. 20, Panel A); K113A had noeffect on fH binding (FIG. 20, Panel A) while D121A decreased fH bindingby about 4-fold compared with binding of fH by wildtype fHbp ID 77 (FIG.20, Panel A). Anti-fHbp JAR 31 bound to all three mutants, whichindicated that respective amino acid substitutions did not affect theepitope recognized by this mAb.

Double amino acid substitutions, R41S/K113A, R41S/K119A and R41S/D121Awere also constructed in fHbp ID 77. The R41S/K119A mutant showed about5-fold decrease in fH binding by ELISA (FIG. 21, Panel A), while theR41S/K113A and R41S/D121 mutant had about 10-fold less binding to fHthan the wildtype fHbp ID 77 (FIG. 21, Panel A and Table 6). Anti-fHbpmAb JAR 31 showed similar binding with all three of these double mutantsof ID 77 and the wildtype fHbp ID 77, which indicated that there weresimilar amounts of each of the fHbp variants adhered to the microtiterwells and that these amino acid substitutions did not affect the epitoperecognized by this mAb.

A triple amino acid substitution R41S/K113A/D121A was introduced in fHbpID 77. This triple mutant exhibited no fH binding (FIG. 22, Panel A).The mutant fHbp retained binding to JAR 31 (FIG. 22, Panel C), buteliminated JAR 4 binding (FIG. 22, Panel B). In contrast, theK113A/D121A double mutant had approximately 10-fold decreased binding offH, which indicated that these substitutions in addition to the R41Ssubstitution contributed to the loss of fH binding.

Example 14. Effect of Amino Acid Substitutions in fHbp ID 22 (ModularGroup III) on Binding of fH

fHbp ID 22 is representative of fHbp sequence variants in modular groupIII (variant group 2, FIG. 16). Mutations were introduced in fHbp ID 22at positions R80, D211, E218, D248, G236 (Table 6), and R41, Q38, A235,Q126, D201 and E202 (Table 7). The fHbp mutants were produced asdescribed above. Specifically, R80A, D211A, E218A, E248A, R41S, Q38A,Q126A, G236I, A235G, D201A, and E202A substitutions were introducedsingly into fHbp ID 22. In addition, T221A/H223A double substitutionswere introduced into fHbp ID 22. The mutants were then characterized forfH binding and binding to anti-fHbp mAbs by ELISA (Tables 6 and 7).

The ability of these mutants to bind to human fH was tested as describedabove in Example 2, and compared to the ability of wild-type fHbp ID 22to bind to human fH. The results are shown in FIG. 23, panels A-C andsummarized in Tables 6 and 7. As shown in FIG. 23 panels A and B, theD211A, R80A, E218A, and E248A substitutions in fHbp ID22 reduced bindingto fH by more than 50-fold compared with binding fH by the wildtype fHbpID 22 (See also Table 6). As shown in FIG. 23 panel C, the R41S, Q38A,and Q126A substitutions did not significantly reduce binding to fH(<4-fold; see also Table 7).

As shown in FIG. 24, panels A and B, the R80A, D211A, E218A, and E248Amutants of fHbp ID22 retained binding to MAb JAR31, indicating that theJAR31 epitope is preserved in each of these mutants.

As shown in FIG. 25, panels A and B, the D211A and the E218A mutants offHbp ID 22 retained binding to MAb JAR4, indicating that the JAR4epitope is preserved in these mutants. As shown in FIG. 25 the R80Amutant did not retain binding to MAb JAR4 (panel A), and the E248Amutant showed reduced binding to MAb JAR4 (panel B).

As shown in FIG. 26, panels A and B, the R80A, D211A, E218A, and E248Amutants of fHbp ID 22 retained binding to MAb JAR35, indicating that theJAR35 epitope is preserved in each of these mutants.

As shown in FIG. 27, panel A, the T221A/H223A double substitution andthe G236I single substitution in fHbp ID 22 reduced binding to human fHby more than 50-fold compared with binding of fH by wildtype fHbp ID 22(See also Table 6). As shown in FIG. 27, the T221A/H223A mutant in fHbpID 22 retained binding to MAb JAR31 (panel B), MAb JAR 35 (panel C), andMAb JAR 4 (panel D), which indicates that the JAR31, JAR35, and JAR4epitopes are preserved in the T221A/H223A mutant. As shown in FIG. 27,the G236I mutant in fHbp ID 22 retained binding to MAb JAR 35 (panel C),but exhibited reduced binding to MAb JAR 31 (panel B), and had little orno binding to JAR 4 (panel D).

As shown in FIG. 28, panel A, the R41S, Q38A, and A235G singlesubstitutions in fHbp ID 22 did not significantly reduce binding tohuman fH. As shown in FIG. 28, the R41S, Q38A, and A235G mutantsretained binding to MAb JAR31 (panel B), and to MAb JAR 35 (panel C),indicating that the JAR31 and JAR35 epitopes are preserved in each ofthe R41S, Q38A, and A235G mutants.

As shown in FIG. 29, panel A, the Q126A, D201A, and E202A singlesubstitutions in fHbp ID 22 did not significantly reduce binding tohuman fH. As shown in FIG. 29, panel B, the Q126A, D201A, and E202Amutants retained binding to MAb JAR35, which indicated that the JAR35epitope is preserved in each of these mutants.

The effect of various single and double amino acid substitutions on theability of fHbp ID 1, ID 22, and ID 77 to bind to human fH, and to bindto various monoclonal antibodies, is summarized in Tables 6 and 7.

TABLE 6 Mutations that decrease fH binding Background fHbp Fold-Decreasein Sequence Variant Amino Acid fH binding Anti-fHbp MAb Reactivity^(¶)(modular group*) Mutation (FIG. Number) JAR5 JAR4 JAR31 JAR35 ID 1 (I)None (WT) 0 2 2 0 0 R41S >50 (F10) 2 2 NA NA R41A >50 (F10) 2 Not NA NADone R130A >50 (F15) 2 1 NA NA H119A >10 (F15) 2 1 NA NA E218A >50^(†)(F4) 2 2 NA NA E239A >10^(†) (F4) 2 2 NA NA E218A/E239A >50^(†) (F4) 2 2NA NA ID 4 (I) R41S >50 (F11) 2 2 NA NA ID 9 (I) R41S >50 (F11) 2 2 NANA ID 74 (I) R41S >50 (F11) 2 2 NA NA ID 15 (IV) None (WT) 0 2 0 0 0S41P >50 (F17) 2 NA 0 NA ID 22 (III) None (WT) 0 0  2^(†) 2 2 R80A >50(F23) NA 0 2 2 D211A >50 (F23) NA 2 2 2 E218A >50 (F23) NA 2 2 2E248A >50 (F23) NA 1 2 2 G236I >50 (F27) NA 0 1 2 T220A/H222A >50 (F27)NA 2 2 2 ID 77 (VI) None (WT) 0 0  2^(†) 2 0 R41S/K113A >10 (F21) NA 1 2NA R41S/K119A >5 (F21) NA 1 2 NA R41S/D121A >10 (F21) NA 1 2 NAR41S/K113A/D121A >50 (F22) NA 0 2 NA *Modular group based on lineages offive variable segments, see FIG. 16. Modular group I and IV are in theantigenic variant 1 group; modular groups III and VI are in antigenicvariant group 2. ^(¶)Compared with binding of mAb to respective wildtypesequence variant; 0, no significant binding by MAb; 1, diminishedbinding (>30% decrease), 2, similar or higher binding (<30% decrease).^(†)JAR 4 binds about 30% less to variant 2 fHbp (i.e., ID 22 or 77)than to variant 1 (i.e., ID 1) ^(†)FIG. 5 of U.S. Patent Publication No.2006/0029621 **NA, not applicable; for mAb reactivity, the antibody doesnot bind to respective wildtype sequence variant

TABLE 7 Mutations that do not significantly decrease fH bindingBackground fHbp Fold-Decrease in Sequence Variant Amino Acid fH binding*Anti-fHbp MAbs (Modular Group*) Mutation (FIG. No.) JAR5 JAR4 JAR31JAR35 ID 1 (I) None (wildtype) 0 2 2 0 0 K241E 0 (F14) 2 2 NA NA Q87A 02 2 NA NA Q113A 0 2 2 NA NA I114A/S117A 0 2 2 NA NA G121R 0 2 2 NA NA ID15 (IV) None (Wildtype)_(—) 0 2 0 0 0 E241K 0 (F14) 2 NA NA NA ID 19(VI) None (wildtype) 0 0 2 R41S 0 (F12) NA 1 2 NA ID 22 (III) Q38A 0(F28) NA 1 2 2 R41S 0 (F28) NA 1 2 2 A235G 0 (F28) NA 1 2 2 Q126A 0(F29) NA 2 2 2 D201A 0 (F29) NA 1 1 2 E202A 0 (F29) NA 1 2 2 ID 77 (VI)R41S 0 (F12) NA   ND*** 2 NA K113A 0 (F20) NA ND 2 NA K119A 0 (F20) NAND 2 NA D121A 0 (F20) NA ND 2 NA *Modular group based on lineages offive variable segments, see FIG. 16. Modular group I and IV are in theantigenic variant 1 group; modular groups III and VI are in antigenicvariant group 2. *Compared with fH binding by respective wildtype fHbpvariant. ^(¶)Compared with binding to respective wildtype sequencevariant; 0, no significant binding by mAb; 1, diminished binding (>30%decrease), 2, similar or higher binding (<30% decrease) **NA, notapplicable; mAb does not bind to respective wildtype sequence variant***ND, not tested

As shown in FIG. 30, panel A, the E218A single substitutions in fHbp ID28 reduced binding to human fH compared with binding of fH by wildtypefHbp ID 28. Also as shown in FIG. 30, panel A, the E197A, K245A, andD201A single substitutions in fHbp ID 28 did not significantly reducebinding to fH. FIG. 30, panel B shows binding of mouse polyclonalanti-fHbp ID28 antiserum to the various proteins (WT fHbp; and E197A,K245A, and D201A single substitutions in fHbp ID 28). The data presentedin FIG. 30, panel B indicate that the various fHpb are present on theELISA plate in similar quantities. As shown in FIG. 30, panels C and D,the E218A mutant bound to JAR 31 and JAR 33 MAbs, indicating that theoverall conformations of the epitopes recognized by these MAbs areretained.

The overall immunogenicity of the fHbp mutants can be determined byadministering the mutants as vaccines to wildtype mice whose native fHdoes not bind to the mutant or wildtype vaccines. The data generated inthis model provide an overall assessment of whether or not the epitopesimportant in eliciting serum bactericidal antibodies are retained in themutant vaccine. For example, the E218A/E239A mutant in fHbp ID 1eliminated binding with human fH but in multiple studies had impairedability to elicit bactericidal antibody responses in WT mice (Table 2,above). The immunogenicity experiments are carried out as describedabove in Example 1. The titers of IgG and bactericidal antibodies aremeasured and compared to the corresponding levels found in miceadministered with the corresponding wild-type and/or negative controls.If the critical epitopes needed for eliciting bactericidal activity areretained by the mutant vaccine, the expectation is that the levels ofantibody elicited in the wildtype mice will not be significantlydifferent than the levels elicited by the corresponding wild-type fHbpvaccine.

Example 15. Induction of Bactericidal Response by fHbp Variants

Wildtype BALB/c mice (whose fH does not bind to the WT fHbp) wereimmunized intraperitoneally with three doses of recombinant fHbpvaccines, with each dose separated by three-week intervals. Each dosecontained 10 μg of recombinant fHbp and 300 μg Al(OH)₃ in a volume of100 μl (final buffer composition was 10 mM Histidine, 150 mM NaCl, pH6.5). Blood samples were obtained by cardiac puncture three weeks afterthe third dose.

Serum bactericidal titers were measured against group B strain H44/76(ID 1) or group W-135 strain Ghana 04/07 (ID 22) using IgG depletedhuman complement (Beernink et al, J Immunology 2011). Not Different,geometric mean titers (GMTs) between mutant and respective WT vaccinewere not significantly different (P>0.10 by T test on log 10 transformedtiters).

The data are shown in FIGS. 31-33. FIG. 31 shows serum bactericidaltiters of mice immunized with mutants of fHbp ID 1 vaccines withdecreased binding with human fH. Each symbol represents the titer of anindividual mouse measured against group B strain H44/76 (ID 1).Horizontal lines represent geometric mean titers. The respective GMTs ofeach of the mutant vaccines were not significantly different than thatelicited by the WT fHbp ID 1 vaccine (P>0.10).

FIG. 32 shows serum bactericidal titers of mice immunized with mutantfHbp ID 22 vaccines with decreased binding with human fH. Each symbolrepresents the bactericidal titer of an individual mouse measuredagainst group W-135 strain Ghana 7/04 (ID 22). Horizontal linesrepresent geometric mean titers. Upper panel. Mutant vaccines (D211A,E218A, E248A and T221A/H223A) with GMTs that were not significantlydifferent than that of WT ID 22 vaccine (P>0.10). Lower panel, Mutantvaccines (R80A and G236I) that elicited significantly lower GMTs thanthat of WT ID 22 vaccine (P<0.05).

FIG. 33 shows serum bactericidal titers of mice immunized with mutantfHbp ID 77 vaccine with decreased binding with human fH. Each symbolrepresents the bactericidal titer of an individual mouse measuredagainst group W-135 strain Ghana 7/04 (ID 22). Horizontal linesrepresent geometric mean titers. Mice immunized with the tripleR41S/K113A/D121A mutant ID 77 vaccine had a significantly lower GMT thanmice immunized with WT vaccine (P<0.05).

Table 8 summarizes the immunogenicity data shown in FIGS. 31-33. NotDifferent, geometric mean titers (GMTs) between mutant and respective WTvaccine were not significantly different (P>0.10 by T test on log 10transformed titers).

TABLE 8 Immunogenicity of fHbp mutants with decreased fH bindingBactericidal Activity fHbp No of Titers vs. fHbp ID Vaccine Mice StrainRespective WT 1 WT 14 H44/76 n/a 1 R41S 14 H44/76 Not Different 1 R41A14 H44/76 Not Different 1 R130A 12 H44/76 Not Different 1 E239A 12H44/76 Not Different 22 WT 10 Ghana 04/07 n/a 22 D211A 10 Ghana 04/07Not Different 22 E218A 10 Ghana 04/07 Not Different 22 E248A 10 Ghana04/07 Not Different 22 T221A/H223A 10 Ghana 04/07 Not Different 22 R80A10 Ghana 04/07 Lower 22 G236I 10 Ghana 04/07 Lower 77 WT 12 Ghana 04/07n/a 77 R41S/K113A/D121A 12 Ghana 04/07 Lower

Example 16. Sequence Alignments

FIGS. 34 and 35 present an amino acid sequence alignments of fHbp ID 1(SEQ ID NO:1), fHbp ID 22 (SEQ ID NO:2), fHbp ID 77 (SEQ ID NO:4), fHbpID 28 (SEQ ID NO:3), and ID1/ID77 chimera (SEQ ID NO:8) amino acidsequences. ID 28 is shown as a reference sequence for fHbp variant group3. Factor H binding interface residues (highlighted in gray) are asdescribed in Schneider et al. ((2009) Nature 458:890-3) described ashydrogen bond or ionic interactions. GEHT (SEQ ID NO:27) at position 136to 139 represents the junction point between ID 1 and ID 77 for thechimeric fHbp.

FIG. 35. Alignment of fHbp ID 1 (SEQ ID NO:1), fHbp ID 22 (SEQ ID NO:2),fHbp ID 77 (SEQ ID NO:4), fHbp ID 28 (SEQ ID NO:3), and ID1/ID77 chimera(SEQ ID NO:8) amino acid sequences. Residues highlighted in grayindicate residues mutated and summarized in Table 7.

Table 9, below, summarizes MAb reactivity of fHbp ID 1, ID 22, ID 77,and ID 28.

TABLE 9 ID Variant Modular Group MAb Reactivity 1 1 I JAR 4, JAR 5 22 2III JAR 4, JAR 31, JAR 35 77 2 VI JAR 4, JAR 31, JAR 35 28 3 II JAR 31,JAR 33

Example 17. Efficacy of Inhibition of fH/fHbp Binding Correlates withBactericidal Activity; and the Role of NspA

Materials and Methods

Murine Anti-fHbp mAbs.

The hybridoma cell lines producing murine fHbp-specific monoclonalantibodies (mAbs) JAR 3 (IgG3), JAR 5 (IgG2b) and mAb502 (IgG2a;Giuliani et al. (2005) Infect. Immun. 73:1151; and Scarselli et al.(2009) J. Mol. Biol. 386:97) were used. Control mAbs included SEAM 12(Granoff et al. (1998) J. Immunol. 160:5028), which reacts with thegroup B capsule, and an anti-PorA P1.7 (NIBSC code 01/514, obtained fromthe National Institute for Biological Standards and Control, PottersBar, United Kingdom).

Human IgG1 Chimeric Mouse Anti-fHbp mAbs.

RNA isolated from the hybridoma cells was converted into cDNA using anOmniscript RT Kit (Qiagen), according to the manufacturer'sinstructions. cDNA was amplified using immunoglobulin heavy (H) andlight (L) chain-specific primers (Wang et al. (2000) Infect. Immun.68:1871) and inserted into the pGem vector (Promega) for sequencing.Based on the determined sequences, specific primers were designed tofacilitate the insertion of the murine VH and VL sequences into amodified FRT bicistronic eukaryotic expression vector (Invitrogen). Foreach antibody, the murine VL sequence was inserted downstream of a humankappa L chain leader sequence, and in frame with a human kappa L chainconstant sequence. The murine VH sequence was inserted downstream from ahuman H chain leader sequence, and in frame with a complete human IgG1constant region sequence. The vector utilized an Internal RibosomalEntry Segment (IRES) sequence between the VH and VL sequences tofacilitate balanced translation of both chains. The DNA sequences of allconstructs were verified prior to transfection.

Flp-In CHO cells (Invitrogen) were plated at 3.5×10⁵ cells per well (in2 mL Flp-In medium) in Nunclon Delta 6-well plates and then incubated at37° C., 5% CO₂ overnight. Once cells reached 80% confluence they weretransfected with pOG44 and the FRT vector containing the VH and VLinserts (9:1 ratio) using the TransFast transfection reagent (Promega).Forty-eight hours after transfection, the cells were trypsinized andplaced in a fresh 6-well plate under drug selection with 600 μg/mlhygromycin. Transfected cells were adapted to serum-free suspensionculture using Excell 302 medium (Sigma Aldrich), and grown forapproximately 2 weeks. Antibody from the cell culture supernatant wasconcentrated prior to purification using a 200 ml stirred cell (Amicon)and applying nitrogen gas pressure. Antibody was purified using HiTrapprotein G columns (GE Healthcare) followed by extensive dialysis againstPBS. BSA was added to a final concentration of 1% and aliquots werestored to −30° C.

ELISA.

Concentrations of the human IgG1-mouse chimeric mAbs were determined bya capture ELISA with goat anti-human κ chain specific antibody(Biosource) bound to wells of a microtiter plate. Bound human IgG wasdetected by goat anti-human IgG antibody conjugated with alkalinephosphatase (Invitrogen). Antibody concentrations were assigned bycomparison with concentration-dependent binding of a human IgG1 standard(monoclonal κ chain antibody from human myeloma, Sigma). Binding of theanti-fHbp mAbs to fHbp was measured by ELISA with recombinant fHbp onthe plate. The secondary detecting antibody was goat anti-human κ chainspecific antibody conjugated with alkaline phosphatase (Biosource).

Surface Plasmon Resonance.

The kinetics of binding of the human-mouse chimeric mAbs to fHbp wasmeasured by surface plasmon resonance with immobilized recombinant fHbp(500 or 1000 response units) on CMS chips (GE Healthcare, Piscataway,N.J.), which was performed via amine coupling (Amine Coupling kit, GEHealthcare). Kinetics of binding were determined at mAb concentrationsranging from 0.016 to 50 μg/ml (0.1 μM to 333 μM) using a Biacore T/100instrument (GE Healthcare, Piscataway, N.J.).

Binding to N. meningitidis by Flow Cytometry.

Binding of the chimeric mAbs to the surface of live encapsulatedbacteria was measured with strain H44/76 (B:15:P1.7,16; ST -32), whichis a relatively high expresser of fHbp ID 1 (Welsch et al. (2008) J.Infect. Dis. 197:1053; Welsch et al. (2004) J. Immunol. 172:5606). Insome experiments, isogenic knockout (KO) mutants of H44/76 in whichfHbp, NspA or both proteins were not expressed, were tested. Therespective genotypes were fHbp:Erm, NspA:Spc, and fHbp:Erm/NspA:Spc(Lewis et al. (2010) PLoS Pathog. 6:e1001027). The binding assay wasperformed as previously described except that test or control antibodieswere incubated together with the cells for 1 hr at room temperatureinstead of 2 hrs on ice. Antibody bound to the bacteria was detected byCF488A goat anti-human IgG (BioTium).

Inhibition of Binding of fH.

The ability of the anti-fHbp mAbs to inhibit binding of fH to fHbp wasmeasured by ELISA. Wells of a microtiter plate were coated withrecombinant fHbp (2 μg/ml). Dilutions of the mAbs were added andincubated at 37° C. for 2 hrs, followed by human fH (Complement Tech.),2 μg/ml, which was incubated for an additional 1 hour at roomtemperature. After washing with PBS-0.05% Tween 20, bound fH wasdetected by a sheep polyclonal antiserum to fH (Abcam) followed bydonkey anti-sheep IgG antibody (Sigma Aldrich) conjugated with alkalinephosphatase. The results were expressed as the percentage of inhibitionof fH binding in the presence of an anti-fHbp mAb compared with fHbinding in the absence of the mAb.

The ability of the anti-fHbp mAbs to inhibit binding of fH to thesurface of live bacterial cells was measured by flow cytometry. H44/76bacteria were incubated with 50 μg/ml of anti-fHbp mAb and differentconcentrations of purified fH for 30 mins at room temperature. Afterwashing the cells, bound fH was detected by a sheep polyclonal antiserumto fH (Lifespan Bioscience) followed by donkey anti-sheep IgG antibody(Invitrogen) conjugated with green-fluorescent Alexa Fluor 488 dye. Insome experiments 20% IgG-depleted human serum, which contained 90 μg/mlof fH, was used as the source of human fH. To avoid bacteriolysis, thehuman serum was heated for 30 mins at 56° C. to inactivate heat-labilecomplement components essential for bacteriolysis. This heat treatmentdid not affect fH activity.

Human Complement Sources.

The primary complement source for measurement of bactericidal activityand C4b deposition was serum from a healthy adult with normal totalhemolytic complement activity and no detectable serum bactericidalantibodies against the test strain. To eliminate non-bactericidal IgGantibodies, which might augment or inhibit the activity of the testmAbs, the complement source was depleted of IgG using a protein G column(HiTrap Protein G, GE Life Sciences, Piscataway, N.J.). A 1-ml aliquotof human serum was passed over a protein G column (1 ml HiTrap ProteinG, GE Life Sciences, Piscataway, N.J.) equilibrated with PBS and theflow-through fraction was collected. Adequacy of IgG depletion wasmonitored by an IgG capture ELISA, and CH50 activity was assayed with acommercial assay (EZ Complement CH50 test kit, Diamedix Corp., Miami,Fla.). To avoid bacteriolysis when measuring C4b deposition, thecomplement source was depleted of C6 using an anti-human complement C6affinity column, as previously described (Plested and Granoff (2008)Clin. Vaccine Immunol. 15:799). In some experiments, commercial humancomplement sources that had been depleted of fH or C1q (ComplementTech.), which was also depleted of IgG as described above, were used.

Serum Bactericidal Assay.

Bactericidal activity was measured as previously described (Beernink etal. (2009) J. Infect. Dis. 199:1360) using log-phase bacteria of group Bstrain H44/76 and 20% human serum depleted of IgG as a complementsource. Immediately before performing the assay, the anti-fHbp mAbs werecentrifuged for two hours at 100,000×g to remove aggregates.Bactericidal activity (BC_(50%)) was defined by the mAb concentrationthat resulted in a 50% decrease in CFU/ml after 60-min incubation in thereaction mixture compared with the CFU/ml in negative control wells attime zero.

C1q-Dependent, C4b Deposition on N. meningitidis.

Flow cytometry was used to measure deposition of human C4b on thesurface of live bacteria of group B strain H44/76. The bacteria weregrown as described above for the bactericidal assay and were incubatedwith 5% C1q-depleted human serum (Complement Tech.) that had also beendepleted of complement C6 to avoid bacteriolysis (See above). Differentconcentrations of the chimeric human-mouse anti-fHbp mAbs were addedwith or without 20 μg/ml of purified C1q protein (Complement Tech.).Human C4b bound to bacteria was detected with fluorescenceisothiocyanate-conjugated anti-human C4b (Meridian Life Science).

Results

JAR 3 and JAR 5 mAbs inhibit binding of each other to fHbp, andrecognize overlapping epitopes that involve glysine and lysine atpositions 121 and 122, respectively. The respective epitopes recognizedby the two paratopes were differentiated by dissimilar binding byWestern blot with different fHbp amino acid sequence variants. Themurine hybridomas JAR 3 and JAR 5 were derived from the same VH and VLgermline genes, but differed in sequence in their respective CDR regions(with the exception of VL CDR2). The murine mAb502 was encoded bydifferent VH and VL germline genes than those of JAR 3 or JAR 5. mAb502recognizes a conformational epitope requiring an arginine at position204, and does not inhibit binding of JAR 3 or JAR 5 to fHbp. Thus,mAb502 recognizes an fHbp epitope distinct from those recognized by theJAR mAbs. The Genbank accession numbers for vL and vH genes of mAb502are EU835941 and EU835942, respectively. The GenBank accession numbersfor VL and VH regions of JAR3 and JAR5 antibodies are as follows:JF715928, JAR3 variable heavy chain; JF715929, JAR3 variable lightchain; JF715926, JAR5 variable heavy chain; and JF715927, JAR5 variablelight chain.

FIG. 36. Model of fHbp in a complex with a fragment of fH, based on thecoordinates of the crystal structure (Schneider et al. (2009) Nature458:890). Spatial relationship of the amino acid residues previouslyreported to affect binding of anti-fHbp mAb502, and JAR 3 and JAR 5 tofHbp fH fragment, light gray, is shown in complex with fHbp.

The Three Human IgG1 Mouse Chimeric Anti fHbp mAbs have Similar BindingCharacteristics.

Three human-mouse chimeric anti-fHbp antibodies were constructed, inwhich each of the JAR 3, JAR 5 and mAb502 paratopes were paired with ahuman IgG1 constant region. In an ELISA with recombinant fHbp adsorbedto the wells of a microtiter plate, the three mAbs showed similarrespective binding (FIG. 37, Panel A). By surface plasmon resonance, therespective kinetics of binding with 200, 500 or 1000 RU of immobilizedfHbp were similar for the three mAbs, which were each tested atconcentrations from 0.016 to 2.25 μg/ml. Representative data for 0.25μg/ml (1.7 μM) of mAb and 1000 RU of immobilized fHbp ID 1 are shown inPanel B. Although mAb502 showed lower peak binding to fHbp than JAR 3 orJAR 5, the respective association rates, K_(a), were similar (4.25E+06,2.26E+06 and 1.19E+06, for JAR 3, JAR 5 and mAb 502, respectively). Thedissociation rates were slow for all three mAbs, which precludedcalculation of accurate dissociation constants. The order of magnitudeof the K_(d) values for each of the mAbs was E -05.

mAb binding to the surface of live bacteria of group B strain H44/76 wasmeasured by indirect fluorescence flow cytometry. At mAb concentrationsbetween 0.8 and 40 μg/ml, the respective binding of the three mAbs wasindistinguishable from each other. The binding results obtained with 4μg/ml are shown in FIG. 37, Panel C. Binding was not affected by thepresence of heat-inactivated 20% IgG-depleted human serum, whichcontained ˜90 μg/ml of human fH (Compare FIG. 37, Panel D with FIG. 37,Panel C).

FIGS. 37-D. Binding of fHbp-specific mAbs to fHbp. A. ELISA. Bound IgGwas detected with an anti-human kappa light chain-specific alkalinephosphatase conjugated antibody. B. Surface plasmon resonance. Bindingof anti-fHbp mAbs (0.25 μg/ml) to immobilized recombinant fHbp (1000RU). C. Flow cytometry. Binding of anti-fHbp mAbs (4 μg/ml) with livebacterial cells of N. meningitidis group B strain H44/76. JAR 3, blackdotted line; JAR 5, gray line; mAb502, black line. An irrelevant mAb(100 μg/ml) was a negative control (gray filled histogram). The bindingcurves of the three anti-fHbp mAbs are superimposed. D. Flow cytometry.Same mAb concentrations as in Panel C with the addition of 20%IgG-depleted human serum as a source of human fH (˜90 μg/ml).

All Three Human-Mouse Chimeric mAbs Activate the Human ClassicalComplement Pathway but Only JAR 3 and JAR 5 have Bactericidal Activity.

Activation of the classical complement pathway is initiated when IgGbinds to the bacterial surface and there is sufficient antigen-antibodycomplex to allow proximate Fc regions of the antibody to engage C1q,which in turn activates C4b. C4b binding to the surface of live N.meningitidis cells of group B strain H44/76 was measured as a surrogatefor C1q binding and C4b activation, and as a marker for classicalcomplement pathway activation.

When the source of complement was 5% C1q-depleted human serum that hadalso been depleted of IgG, there was negligible C4b deposition elicitedby the anti-fHbp mAbs (FIG. 38, Panel A). When 20 μg/ml of purified C1qwas added back to the reaction mixture, each of the mAbs activated C4bdeposition (FIG. 38, Panel B). Although binding of each of the mAbsactivated the classical complement pathway only JAR 3 and JAR 5 hadcomplement-mediated bactericidal activity (FIG. 38, Panel C). The meanconcentrations±SE for 50% killing in three assays were 9±0.85 μg/ml forJAR 3; 15±2 μg/ml for JAR 5 (P=0.024), and >100 μg/ml for mAb502(P<0.0003 compared to JAR 3 or JAR 5).

FIGS. 38A-C. C1q-dependent complement activation on encapsulated group Bbacteria of strain H44/76. A. Flow cytometry. Activation of C4bdeposition by 4 μg/ml of mAb and complement (5% IgG-depleted humanserum) that had been depleted of C1q. Anti-fHbp mAb JAR 3, black dottedline; JAR 5, gray line; mAb502, black line, and an irrelevant human mAb(100 μg/ml; gray filled histogram) (data for each are superimposed). B.Flow cytometry. Same symbols and conditions as in Panel A except for theaddition of 20 μg/ml of purified C1q protein to the reactions. C.Bactericidal activity. Survival of bacteria after incubation for 60 minat 37° C. with each of the mAbs and complement (20% IgG-depleted humanserum).

Chimeric mAbs JAR 3 and JAR 5, but not mAb502, Inhibit Binding of fH.

In previous studies, murine mAbs JAR 3 and JAR 5 inhibited binding of fHto fHbp whereas murine mAb502 did not inhibit fH binding. By ELISA, thehuman IgG1 chimeric JAR 3 and JAR 5 mAbs also inhibited binding of fH tofHbp while the chimeric mAb502 did not inhibit fH binding (FIG. 39,Panel A). When 20% heat-inactivated IgG-depleted human serum was thesource of fH, 50 μg/ml of chimeric JAR 3 or JAR 5, but not mAb502,inhibited binding of fH to the surface of live bacterial cells (FIG. 39,Panel B). As little as 2 μg/ml of JAR 3 or JAR 5 also inhibited bindingof fH (Panel C) although inhibition was less complete than with 50 μg/mlof the mAb (Panel B).

FIGS. 39A-C. Inhibition of fH binding by anti-fHbp mAbs. A. ELISA: fH (4μg/ml) with solid-phase recombinant fHbp. B and C. Flow cytometry, withlive bacterial cells of group B strain H44/76; Light gray filled area,bacteria with fH (˜90 μg/ml) in 20% IgG-depleted human serum without amAb; black solid line, bacteria with serum fH+mAb502, 50 μg/ml; dottedblack line, bacteria with serum fH+JAR 3, 50 μg/ml; gray solid line,bacteria with serum fH+JAR 5, 50 μg/ml; dark gray filled area, bacteriawithout fH or mAb as a negative control. fH binding was detected with anfH-specific sheep antibody. C. Same conditions as in Panel B except that2 μg/ml of each of the anti-fHbp mAbs was tested instead of 50 μg/ml.

The correlation observed between bactericidal activity and mAbinhibition of fH binding does not necessarily prove that inhibition wasresponsible for the greater bactericidal activity of JAR 3 or JAR 5 thanmAb502. For example, the spatial relationships of fHbp epitopes on thesurface of the bacteria that are recognized by anti-fHbp mAbs thatinhibited fH binding are different than those of epitopes recognized byanti-fHbp mAbs that did not inhibit fH binding (compare, for example,the positions of the amino acid residues previously reported to affectbinding of mAb502 (Scarselli et al. (2009) J Mol Biol 386:97-108) withthose of JAR 3 and JAR 5 (Beernink et al. (2008) Infect Immun76:4232-4240)(FIG. 36). These spatial differences could potentiallyaffect the formation of a functional membrane attack complex independentof fH down-regulation.

To determine whether the differences in the locations of the respectiveepitopes affected bactericidal activity independent of fH inhibition,anti-fHbp bactericidal activity was measured with fH-depleted complement(20% human serum that also had been depleted of IgG). In the absence offH, all three mAbs showed similar complement-mediated bactericidalactivity (BC_(50%), 1.2 to 1.4 μg/ml, Table 10). In contrast, whenpurified human fH was added to the reaction mixture, mAb502 was nolonger bactericidal (BC_(50%)>100 μg/ml, Panel B). Adding fH to thereaction mixture also decreased bactericidal activity of two controlmurine mAbs reactive with the group B capsule or PorA (comparerespective BC_(50%) values measured with fH depleted complement, PanelC, with those with fH-repleted complement, Panel C) but the effect of fHrepletion was less pronounced than with the anti-fHbp mAbs.

TABLE 10 Anti-fHbp mAb bactericidal activity measured with fH-depletedhuman complement Bactericidal Activity (BC_(50%), μg/ml)* fH-depletedfH-repleted Complement Complement Mean Range Mean Range Human IgG1chimeric mouse anti-fHbp mAbs JAR 3 1.4 0.8-2.0 15.2 12.5-18   JAR 51.25 1.0-1.5 23.5 22-25 mAb502 1.25 0.75-1.5  >100 >100 Control mouseIgG2a mAbs Anti-PorA P1.7 0.5 0.3-0.7 1.05 1.0-1.1 Anti-capsular, SEAM12 0.18 0.15-0.2  1.15 1.0-1.2 *Data are mean and respective ranges ofthe concentrations of the mAbs that gave 50% killing after 1 hrincubation with complement (BC_(50%)) in two independent assays. For fHrepleted complement, 50 μg/ml of fH was added.

Elimination of fH Binding to NspA Enhances Bactericidal Activity ofAnti-fHbp mAbs JAR 3 and JAR 5, but not mAb502.

The much lower concentrations of anti-fHbp mAbs required forbacteriolyis with fH-depleted than fH-repleted complement suggested thatwhen fH was present, inhibition of fH binding by JAR 3 or JAR 5 wasincomplete (for example, because of binding of fH by a secondmeningococcal ligand such as NspA (Lewis et al. (2010) PLoS Pathog.6:e1001027). To investigate binding of fH independent of binding to thefHbp ligand, fH binding was measured with an isogenic mutant of group Bstrain H44/76 in which the gene encoding fHbp had been inactivated (fHbpKO strain). A second mutant in which both the fHbp and NspA genes hadbeen inactivated served as a control for a possible contributory effectof NspA.

By flow cytometry, the two mutants and the parent strain showed similarrespective binding with a control murine anti-PorA P1.7 mAb (FIG. 40,Panel A). As expected, there was much less binding of fH (100 μg/ml)with the fHbp KO mutant than with the WT strain (compare black line withgray line in FIG. 40, Panel B). In the absence of both fHbp and NspAexpression (dashed line), fH binding was indistinguishable from thenegative control WT bacteria without added fH (light gray filledhistogram). Similar respective results were obtained when 20%IgG-depleted human serum was used as the source of fH (FIG. 40, PanelC).

FIGS. 40A-C. Binding of fH to mutants of group B H44/76 with geneticinactivation of fHbp expression or expression of both fHbp and NspA. A.Anti-PorA mAb (P1.7, 20 μg/ml). Black line, WT; gray line, fHbp KO;dashed line, fHbp KO combined with NspA KO. B. Binding of purified humanfH (100 μg/ml). Designation as in panel A. C. Binding of fH in humanserum (20%, IgG-depleted). Designation as in Panel A. Results werereplicated in two independent assays.

To determine a possible contribution of fH binding to NspA (andcorresponding down-regulation of complement activation) to the highanti-fHbp mAb concentrations required for bacteriolysis in the presenceof fH, anti-fHbp bactericidal activity was measured with an isogenicNspA KO mutant (FIG. 41). With chimeric JAR 3 or JAR 5, which inhibitedbinding of fH to fHbp, there was significantly greater killing of theNspA KO mutant than the control WT strain (FIG. 41, Panels A and B,respectively). In contrast, chimeric mAb502, which did not inhibit fHbinding, had no bactericidal activity against either strain (FIG. 41,Panel C). Two control mouse mAbs, anti-PorA and anti-capsular, showedsimilar respective bactericidal activity against the WT and mutant NspAKO strains (FIG. 41, Panels D and E, respectively).

FIGS. 41A-E. Bactericidal activity of anti-fHbp mAbs measured against amutant of group B H44/76 with genetic inactivation of NspA expression.Survival of bacteria after incubation for 60 min at 37° C. with each ofthe mAbs and 20% IgG-depleted human serum as a complement source. Opentriangles, NspA KO mutant; closed triangles, control WT strain. A.Chimeric JAR 3. B. Chimeric JAR 5. C. Chimeric mAb502. D. Control murineanti-Por A mAb (P 1.7). E. Control murine mAb, SEAM 12, reactive withgroup B capsule. Results are from three independent dilutions of themAbs performed in two experiments. Where indicated, respective survivalfor WT and NspA KO strains incubated at mAb dilution was significantlydifferent (*P≤0.02; **P<0.001).

Importance of Binding fH by fHbp on Anti-NspA mAb Bactericidal Activity.

As noted above, using a NspA KO mutant of group B strain H44/76, thedata showed that in the absence of fH bound to NspA, anti-fHbp mAbs thatinhibited fH binding (JAR 3 or JAR 5) had greater bactericidal activitythan when tested against a wildtype strain with NspA expression. Thereverse experiment was also conducted: an anti-NspA mAb AL12 (Moe et al,Infect. Immun. (2002) 70:6021) was tested against a fHbp knockout mutantof a group A strain (Senegal 1/99). As shown in FIG. 42, the fHbp KOmutant was 50-fold more susceptible to killing by the anti-NspA mAb thanthe WT strain. In contrast, there was no significant enhancedsusceptibility of the fHbp KO mutant to killing by a control mAb to PorAP1.9. Bactericidal activity of the mAbs was measured with humancomplement (IgG-depleted human serum).

Further Data that Inhibition of fH by Anti fHbp Antibodies is Importantfor Bactericidal Activity.

Eight of nine African meningococcal isolates tested were susceptible tobactericidal activity of an antiserum from mice immunized with aprototype native outer membrane vesicle (NOMV) vaccine prepared from amutant of group B strain H44/76 with over-expressed fHbp ID 1 (Table11). In contrast, all nine isolates were resistant to the antiserum frommice immunized with the recombinant fHbp ID 1 vaccine (bactericidaltiters <1:10), and only one of the nine isolates was killed by thecontrol antiserum from mice immunized with the NOMV vaccine from thefHbp KO mutant (X5, titer 1:36). Mixing the NOMV fHbp KO antiserum withthe antiserum to the recombinant fHbp ID 1 vaccine did not increasebactericidal activity against any of the test strains (Table 11). Thus,the anti-fHbp antibodies elicited by the NOMV vaccine withover-expressed fHbp appeared to be responsible for the bactericidalactivity against the isolates with fHbp sequence variants that did notmatched the fHbp ID 1 in the NOMV vaccine. There also was no evidence ofcooperative bactericidal activity between antibodies to fHbp andantibodies to other antigens in the NOMV vaccine.

TABLE 11 Bactericidal activity of sera of mice immunized with a nativeouter membrane vesicle vaccine from group B strain H44/76 withover-expressed fHbp ID 1. NOMV Vaccine 1/Serum Titer Recombinant fHbpVaccine Over- Test Strain 1/Serum Titer expressed (fHbp ID)* HomologousfHbp* fHbp ID 1 fHbp KO fHbp ID 1 A3 (ID 5) <10 <10 <10 132 A14 (ID 5)<10 <10 <10 114 W1 (ID 9) <10 <10 <10 <10 W3 (ID 9) 818 <10 <10 43 X3(ID 74) 12204 <10 <10 574 X5 (ID 74) 4066 <10 36 640 X7 (ID 74) 7680 <10<10 324 *Strains A3 and A14 are capsular group A, W1 and W3 are capsulargroup W-135, and X3, X5 and X7 are capsular group X; All strains wereclinical isolates from patients with meningococcal disease fromSub-Saharan Africa Bactericidal activity (human complement) of storedsera from mice immunized in a previously published study (KoeberlingVaccine 2007, supra) with recombinant fHbp or NOMV vaccines preparedfrom mutants of group B strain H44/76 with over-expressed of fHbp ID 1.Titers are means of the serum dilution for 50% decrease in CFU/ml afterone hr incubation with human complement as measured in at least twoindependent assays. **Titer with respective recombinant fHbp vaccine ID5, 9 or 74 of that of the test strain

The Broad Serum Cross-Reactive Anti fHbp Bactericidal Activity Inducedby the Mutant NOMV Vaccine is Associated with Higher Anti fHbp AntibodyResponses and Greater Blocking of Binding of fH to fHbp than theRecombinant fHbp Vaccine.

By ELISA, the mice immunized with the NOMV vaccine from the mutant withover-expressed fHbp ID 1 had higher serum anti-fHbp ID 1 antibodyconcentrations than mice immunized with the recombinant fHbp ID 1vaccine (respective geometric means of 2203 and 746 U/ml, P<0.02, FIG.43, Panel A). By ELISA, the sera from the mice immunized with the mutantNOMV vaccine also showed greater inhibition of binding of fH to fHbp ID4, which was the sequence variant expressed by group A strains (FIG. 43,Panel B, P<0.05 at each dilution tested). The increased fH inhibitionwas not only a result of the higher serum anti-fHbp concentrations inthe mutant NOMV vaccine group since on average the anti-fHbp antibodyconcentration required for inhibition of fH in this group was nearly4-fold lower than in the recombinant fHbp vaccine ID 1 group (respectivegeometric means of 1.17 vs. 4.04 U/ml, P<0.05, FIG. 43, Panel C).

FIG. 43, Panel A Anti-fHbp antibody responses to vaccination as measuredby ELISA (Panel A), and the ability of serum anti-fHbp antibodies toinhibit binding of fH to fHbp (Panels B and C, also by ELISA). Mice wereimmunized with recombinant fHbp ID 1 vaccine (filled triangles), or NOMVvaccines prepared from mutants of group B strain H44/76 withover-expressed of fHbp ID 1 (open circles) or a fHbp knock-out (filledcircles). For the recombinant fHbp vaccine and the NOMV vaccine withover-expressed fHbp ID 1, each symbol (Panel A and C) represents theresult of an individual mouse (10 mice per vaccine group). For the NOMVfHbp KO vaccine, each symbol represents the results of testing pooledsera from 3 to 4 individual animals. A) Anti-fHbp ID 1 antibodyconcentrations in arbitrary units per ml. NOMV OE vaccine group hadhigher geometric mean concentration (horizontal line) than miceimmunized with the recombinant fHbp vaccine (p=0.02). B) Inhibition ofbinding of fH to fHbp ID 4, which was heterologous to fHbp ID 1 in thevaccines. At all dilutions, the mean inhibitory activity of the groupgiven the NOMV vaccine from the mutant with over-expressed fHbp (opencircles) was higher than the recombinant fHbp vaccine group (filledtriangles; p<0.05). C) Serum anti-fHbp ID 1 antibody concentration for50% inhibition of binding of fH to fHbp ID 4 (96% amino acid identitywith ID 1). The geometric mean anti-fHbp antibody concentration for 50%inhibition of fH binding was lower for the NOMV OE fHbp group (opencircles) than the recombinant fHbp vaccine group (filled triangles,p<0.05).

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A nucleic acid encoding a non-naturally occurringfactor H binding protein (fHbp) comprising an amino acid sequence,wherein the amino acid sequence is at least 95% identical to the entirelength of the amino acid sequence set forth in SEQ ID NO:1 and comprisesthe substitution of the arginine at position 41 with serine.
 2. Thenucleic acid according to claim 1, wherein the non-naturally occurringfHbp differs from the amino acid sequence of SEQ ID NO:1 by 1 to 10amino acids.
 3. A recombinant expression vector comprising the nucleicacid of claim
 1. 4. A recombinant expression vector comprising thenucleic acid of claim
 2. 5. A genetically modified host cell comprisingthe nucleic acid of claim
 1. 6. A genetically modified host cellcomprising the nucleic acid of claim
 2. 7. A genetically modified hostcell comprising the recombinant expression vector of claim
 3. 8. Agenetically modified host cell comprising the recombinant expressionvector of claim
 4. 9. The genetically modified host cell of claim 5,wherein the genetically modified host cell is a mammalian host cell. 10.The genetically modified host cell of claim 5, wherein the geneticallymodified host cell is Escherichia coli (E. coli).
 11. The geneticallymodified host cell of claim 5, wherein the genetically modified hostcell is a yeast cell.
 12. The genetically modified host cell of claim 5,wherein the genetically modified host cell is Neisseria meningitidisbacterium.
 13. The genetically modified host cell of claim 12, whereinthe Neisseria meningitidis bacterium comprises a genetic modificationthat results in decreased or no detectable toxic activity of lipid A.14. The genetically modified host cell of claim 6, wherein thegenetically modified host cell is Neisseria meningitidis bacterium. 15.The genetically modified host cell of claim 14, wherein the Neisseriameningitidis bacterium comprises a genetic modification that results indecreased or no detectable toxic activity of lipid A.
 16. Thegenetically modified host cell of claim 7, wherein the geneticallymodified host cell is Neisseria meningitidis bacterium.
 17. Thegenetically modified host cell of claim 16, wherein the Neisseriameningitidis bacterium comprises a genetic modification that results indecreased or no detectable toxic activity of lipid A.
 18. Thegenetically modified host cell of claim 8, wherein the geneticallymodified host cell is Neisseria meningitidis bacterium.
 19. Thegenetically modified host cell of claim 18, wherein the Neisseriameningitidis bacterium comprises a genetic modification that results indecreased or no detectable toxic activity of lipid A.